Polyclonal libraries

ABSTRACT

The invention is directed to inter alia two related but self-sufficient improvements in conventional display methods. The first improvement provides methods of enriching conventional display libraries for members displaying more than one copy of a polypeptide prior to affinity screening of such libraries with a target of interest. These methods can achieve diverse populations in which the vast majority of members retaining full-length coding sequences encode polypeptides having specific affinity for the target. In a second aspect, the invention provides methods of subcloning nucleic acids encoding displayed polypeptides of enriched libraries from a display vector to an expression vector without the need for clonal isolation of individual members. These methods result in polyclonal libraries of antibodies and other polypeptides for use, e.g., as diagnostic or therapeutic reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

Commonly owned Ser. Nos. 60/044,292, 08/835,159 and 08/832,935 (now U.S.Pat. No. 6,057,098), all filed Apr. 4, 1997 describe related subjectmatter and are incorporated by reference in their entirety for allpurposes.

BACKGROUND OF THE INVENTION

Over recent years, many publications have reported the use ofphage-display technology to produce and screen libraries of polypeptidesfor binding to a selected target. See, e.g, Cwirla et al., Proc. Natl.Acad. Sci. USA 87, 6378-6382 (1990); Devlin et al., Science 249, 404-406(1990), Scott & Smith, Science 249, 386-388 (1990); Ladner et al., U.S.Pat. No. 5,571,698. A basic concept of phage display methods is theestablishment of a physical association between DNA encoding apolypeptide to be screened and the polypeptide. This physicalassociation is provided by the phage particle, which displays apolypeptide as part of a capsid enclosing the phage genome which encodesthe polypeptide. The establishment of a physical association betweenpolypeptides and their genetic material allows simultaneous massscreening of very large numbers of phage bearing different polypeptides.Phage displaying a polypeptide with affinity to a target bind to thetarget and these phage are enriched by affinity screening to the target.The identity of polypeptides displayed from these phage can bedetermined from their respective genomes. Using these methods apolypeptide identified as having a binding affinity for a desired targetcan then be synthesized in bulk by conventional means.

Phage display technology has also been used to produce and screenlibraries of heterodimeric proteins, such as Fab fragments. See e.g.,Garrard et al., Bio/Tech 9, 1373-1377 (1991). Phage display libraries ofFab fragments are produced by expressing one of the component chains asa fusion with a coat protein, as for display of single-chainpolypeptides. The partner antibody chain is expressed in the same cellfrom the same or a different replicon as the first chain, and assemblyoccurs within the cell. Thus, a phage-Fab fragment has one antibodychain fused to a phage coat protein so that it is displayed from theoutersurface of the phage and the other antibody chain is complexed withthe first chain.

In a further expansion of the basic approach, polypeptide libraries havebeen displayed from replicable genetic packages other than phage. Thesereplicable genetic packages include eucaryotic viruses and bacteria. Theprinciples and strategy are closely analogous to those employed forphage, namely, that nucleic acids encoding antibody chains or otherpolypeptides to be displayed are inserted into the genome of the packageto create a fusion protein between the polypetides to be screened and anendogenous protein that is exposed on the cell or viral surface.Expression of the fusion protein and transport to the cell surfaceresult in display of polypeptides from the cell or viral surface.

Although conventional display methods have achieved considerable successin isolating antibodies and other polypeptides with specific binding toselected targets, some inefficiencies and limitations remain. Inconventional methods, many library members bind nonspecifically to thetarget or the solid phase bearing the target and are amplified alongwith specifically bound library members causing poor efficiency at eachround of affinity selection. Not only can this waste time and effort inperforming many rounds of affinity selection, but members bearingpolypeptides having specific affinity are lost at each round. Selectionis generally terminated when sufficient rounds of affinity selectionhave been performed to achieve a significant number of members bearingpolypeptides with affinity for a target even though many nonspecificallybinding members are still present. Clonal isolates are then picked andtested individually to reduce the risk of losing specific-bindingmembers through further rounds of selection. Clonal isolates shown tobind specifically may then be cloned into an expression work for futureanalysis, and, large-scale production. Accordingly, only one or a few oflibrary members bearing polypeptides with specific affinity for thetarget present in the original repertoire are ever isolated.

The present application provides inter alia novel methods that overcomethese inefficiencies and difficulties, and produce new diagnostic andtherapeutic reagents.

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods of producing a multivalentpolypeptide display library. The starting material is a library ofreplicable genetic packages, such as phage. A member of such a libraryis capable of displaying from its outersurface a fusion proteincomprising a polypeptide to be screened and a tag. The fusion protein isencoded by a segment of a genome of the package. The polypeptides varybetween library members, as does the number of copies of the fusionprotein displayed per library member. The tag is the same in differentlibrary members. The library is contacted with a receptor having aspecific affinity for the tag under conditions whereby library membersdisplaying at least two copies of the fusion protein are preferentiallybound to immobilized receptor by multivalent bonds between the receptorand the at least two copies of the tag. Library members bound to theimmobilized receptor are then separated from unbound library members toproduce a sublibrary enriched relative to the library for membersdisplaying at least two copies of the fusion protein. Polypeptides ofparticular interest are antibodies, particularly Fab fragments.Multivalent Fab phage display libraries can be produced as follows. Thestarting material is a library of phage in which a library membercomprises a phage capable of displaying from its outersurface a fusionprotein comprising a phage coat protein, an antibody light or heavychain variable domain, and a tag. In at least some members, the antibodyheavy or light chain is complexed with a partner antibody heavy or lightchain variable domain chain, the complex forming a Fab fragment to bescreened. The fusion protein and/or the partner antibody heavy or lightchain are encoded by segment(s) of the genome of the phage. The numberof copies of the fusion protein and the partner antibody chain displayedper phage vary between library members. The library or a fractionthereof is contacted with a receptor having a specific affinity for thetag under conditions whereby library members displaying at least twocopies of the fusion protein are preferentially bound to immobilizedreceptor by multivalent bonds between the receptor and the at least twocopies of the tag. Library members bound to the receptor are thenseparated from unbound library members to produce an sublibrary enrichedrelative to the library for members displaying at least two copies ofthe fusion protein. An alternative method of producing a multivalent Fabphage display library is as follows. The starting material is a libraryof phage in which a library member comprises a phage capable ofdisplaying from its outersurface a fusion protein comprising a phagecoat protein, and an antibody light or heavy chain variable domain. Atleast in some members, the antibody heavy or light chain is complexedwith a partner antibody heavy or light chain variable domain chain fusedto a tag, the complex forming a Fab fragment to be screened. The fusionprotein and/or the partner antibody heavy or light chain fused to thetag are encoded by segment(s) of the genome of the phage. The number ofcopies of the fusion protein and the partner antibody chain displayedper phage vary between library members. The library or a fractionthereof is contacted with a receptor having a specific affinity for thetag under conditions whereby library members displaying at least twocopies of the partner antibody chain are preferentially bound toimmobilized receptor by multivalent bonds between the immobilizedreceptor and the at least two copies of the tag. Bound library membersare separated from unbound library members to produce an sublibraryenriched relative to the library for members displaying at least twocopies of the partner antibody chain.

Having produced a polyvalent phage display library, such as describedabove, it can be screened by contacting the library with a targetlacking specific affinity for the tag moiety(ies) and separating librarymembers bound to the target via their displayed polypeptides fromunbound library members. DNA segments encoding polypeptides havingspecific affinity for a target can be subcloned in an expression vector,and the polypeptides expressed in host cells. Polypeptides can then, forexample, be formulated with diagnostic or therapeutic excipients.

In another aspect, the invention provides libraries of nucleic acidsegments encoding polyclonal polypeptides having specific affinity for atarget. Such a library comprises least four different nucleic acidsegments. At least 90% of segments in the library encode polypeptidesshowing specific affinity for a target and no library member constitutesmore than 50% of the library. In some libraries, at least 95% of librarymembers encode polypeptides having specific affinity for a target and nomember constitutes more than 25% of the library. Some libraries have atleast 100 different members. In some libraries, the segments arecontained in a vector. In some libraries, the segment encode antibodychains. In some libraries, first and second segments are present,respectively encoding antibody heavy chains and partner antibody lightchains, which can complex to form a form a Fab fragment. The first andsecond segments can be incorporated into the same or different vectors.

The invention further provides cell libraries in which a member cellcontains a nucleic acid segment from a nucleic acid library, asdescribed above. Such a library of cells can be propagated underconditions in which the DNA segments are expressed to produce polyclonalpolypeptides.

The invention further provides methods of producing polyclonalpolypeptides having specific affinity for a target. The startingmaterial for such methods is a library of replicable genetic packages. Amember comprises a replicable genetic package capable of displaying apolypeptide to be screened encoded by a genome of the package. Thepolypeptides vary between members. DNA encoding at least four differentpolypeptides of the library of replicable genetic packages is subclonedinto an expression vector to produce modified forms of the expressionvector. The modified forms of the expression vector are introduced intoa host and expressed in the host producing at least four differentpolypeptides. At least 75% of modified forms of the expression vectorencode polypeptides having specific affinity for a target and nomodified form of the expression vector constitutes more than 50% of thetotal.

Polypeptides of particular interest are antibodies and these aretypically displayed from a phage libraries. A typical member of such alibrary is a phage capable of displaying from its outersurface anantibody comprising an antibody heavy chain variable domain complexedwith an antibody light chain variable domain. Either the heavy or lightchain variable domain is expressed as a fusion protein with a coatprotein of the phage and either the heavy or light chain variable domainor both is/are encoded by the genome of the phage. The heavy and/orlight chain varies between members. DNA encoding the heavy and/or lightchain variable domains are subcloned from the phage library members intoan expression vector to produce modified forms of the expression vector.The modified forms of the expression vector are introduced into a hostand expressed to produced antibodies formed by the heavy and light chainvariable domains of the phage library in the host. The antibodies arethen released from the host to form an antibody library of at least fourantibodies. At least 75% of modified forms of the expression vectorencode antibodies with specific affinity for a target and no modifiedform of the expression vector constitutes more than 50% of the total.

Polyclonal libraries of antibodies and other polypeptides produced bythe above methods can be incorporated into a diagnostic kit, orformulated for use as a diagnostic or therapeutic reagent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Primers used for amplification of immunoglobulin heavy chains(SEQ ID NOS:20-52).

FIG. 2: Primers used for amplification of immunoglobulin light chains(SEQ ID NOS:53-83).

FIG. 3: Vectors used for cloning antibodies. FIG. 3A shows a vectorobtained from Ixsys, Inc. and described in Huse, WO 92/06204, whichprovides the starting material for producing the vectors shown in FIGS.3B and 3C. FIGS. 3B and 3C show the BS39 and BS45 vectors used in thepresent examples. The following abbreviations are used:

A. Nonessential DNA sequence later deleted.

B. Lac promoter and ribosome binding site.

C. Pectate lyase signal sequence.

D. Kappa chain variable region.

E. Kappa chain constant region.

F. DNA sequence separating kappa and heavy chain, includes ribosomebinding site for heavy chain.

G. Alkaline phosphatase signal sequence.

H. Heavy chain variable region.

I. Heavy chain constant region including 5 amino acids of the hingeregion.

J. Decapeptide DNA sequence.

K. Pseudo gene VIII sequence with amber stop codon at 5′ end.

L. Nonessential DNA sequence that was later deleted.

M. Deleted kappa chain variable sequence with translational stepsequences.

N. Polyhistidine (6 codons_sequence.

O. Same as F above, but lacking the HindIII site.

P. Deleted heavy chain variable sequence with translational stopsequence.

Q. Pseudo gene VIII sequence without amber stop codon at 5′ end.

R. Deleted kappa chain variable sequence with transcriptional stopsequence.

FIG. 4: Oligonucleotides used in vector construction (SEQ ID NOS:84-85).

FIG. 5: Insertion of araC into pBR-based vector (FIG. 5A) and theresulting vector pBRnco (FIG. 5B).

FIG. 6: Subcloning of a DNA segment encoding a Fab by T4 exonucleasedigestion.

FIG. 7: Map of the vector pBRncoH3.

DEFINITIONS

Specific binding between an antibody or other binding agent and anantigen means a binding affinity of at least 10⁶ M⁻¹. Preferred bindingagents bind with affinities of at least about 10⁷ M⁻¹, and preferably10⁸ M⁻¹ to 10⁹ M⁻¹ or 10¹⁰ M⁻¹.

The term epitope means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 Kda). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarily determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. CDR and FRresidues are delineated according to the standard sequence definition ofKabat et al., supra. An alternative structural definition has beenproposed by Chothia et al., J. Mol. Biol. 196, 901-917 (1987); Nature342, 878-883 (1989); and J. Mol. Biol. 186, 651-663 (1989).

The term antibody is used to mean whole antibodies and binding fragmentsthereof. Binding fragments include single chain fragments, Fv fragmentsand Fab fragments The term Fab fragment is sometimes used in the art tomean the binding fragment resulting from papain cleavage of an intactantibody. The terms Fab′ and F(ab′)₂ are sometimes used in the art torefer to binding fragments of intact antibodies generated by pepsincleavage. Here, Fab is used to refer generically to double chain bindingfragments of intact antibodies having at least substantially completelight and heavy chain variable domains sufficient for antigen-specificbindings, and parts of the light and heavy chain constant regionssufficient to maintain association of the light and heavy chains.Usually, Fab fragments are formed by complexing a full-length orsubstantially full-length light chain with a heavy chain comprising thevariable domain and at least the CH1 domain of the constant region.

An isolated species or population of species means an object species(e.g., binding polypeptides of the invention) that is the predominantspecies present (i.e., on a molar basis it is more abundant than otherspecies in the composition). Preferably, an isolated species comprisesat least about 50, 80 or 90 percent (on a molar basis) of allmacromolecular species present. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods). A targetis any molecule for which it is desired to isolate partners withspecific binding affinity for the target.

Targets of interest include antibodies, including antiidiotypicantibodies and autoantibodies present in autoimmune diseases, such asdiabetes, multiple sclerosis and rheumatoid arthritis. Other targets ofinterest are growth factor receptors (e.g., FGFR, PDGFR, EFG, NGFR, andVEGF) and their ligands. Other targets are G-protein receptors andinclude substance K receptor, the angiotensin receptor, the α- andβ-adrenergic receptors, the serotonin receptors, and PAF receptor. See,e.g., Gilman, Ann. Rev. Biochem. 56, 625-649 (1987). Other targetsinclude ion channels (e.g., calcium, sodium, potassium channels),muscarinic receptors, acetylcholine receptors, GABA receptors, glutamatereceptors, and dopamine receptors (see Harpold, U.S. Pat. Nos. 5,401,629and 5,436,128). Other targets are adhesion proteins such as integrins,selectins, and immunoglobulin superfamily members (see Springer, Nature346, 425-433 (1990). Osborn, Cell 62, 3 (1990); Hynes, Cell 69, 11(1992)). Other targets are cytokines, such as interleukins IL-1 throughIL-13, tumor necrosis factors α & β, interferons α, β and γ, tumorgrowth factor Beta (TGF-β), colony stimulating factor (CSF) andgranulocyte monocyte colony stimulating factor (GM-CSF). See HumanCytokines: Handbook for Basic & Clinical Research (Aggrawal et al. eds.,Blackwell Scientific, Boston, Mass. 1991). Other targets are hormones,enzymes, and intracellular and intercellular messengers, such as, adenylcyclase, guanyl cyclase, and phospholipase C. Drugs are also targets ofinterest. Target molecules can be human, mammalian or bacterial. Othertargets are antigens, such as proteins, glycoproteins and carbohydratesfrom microbial pathogens, both viral and bacterial, and tumors. Stillother targets are described in U.S. Pat. No. 4,366,241.

Display library members having full-length polypeptide coding sequenceshave coding sequences the same length as that of the coding sequencesoriginally inserted into a display vector before propagation of thevector.

DETAILED DESCRIPTION I. General

The present invention is directed to inter alia two related butself-sufficient improvements in conventional display methods. The firstimprovement provides methods of enriching conventional display librariesfor members displaying more than one copy of a polypeptide prior toaffinity screening of such libraries with a target of interest. Althoughpractice of the claimed methods is not dependent on an understanding ofmechanism, the rationale for these methods is believed to be thataffinity selection of library members to immobilized binding partnersoccurs predominantly or exclusively through formation of multivalentbonds between multiple copies of displayed polypeptides on a librarymember and immobilized binding partners. Accordingly, only members oflibrary displaying multiple copies of a polypeptide are capable ofsurviving affinity selection to immobilized binding partners. Forexample, conventional libraries of polypeptides fused to pVIII of afilamentous phage typically have Poisson distribution, in which mostmembers display no copies of a polypeptide, a small proportion displayone copy of a polypeptides, a still smaller proportion display twocopies, and a still smaller proportion display three or more copies. Themethods of the present invention enrich for the small proportion ofconventional display libraries displaying two or more copies of apolypeptide. It is this rare fraction of conventional libraries that iscapable of being affinity selected by immobilized binding partners.

Enrichment can be achieved by the inclusion of a tag as a component ofthe fusion protein from which polypeptides are displayed. The tag can beany polypeptide with a known receptor showing high binding specificityfor the tag. The same tag is included in each member of the library.Enrichment is effected by screening the library for affinity binding toan immobilized receptor for the tag. Only library members having two ormore copies of the tag are capable of binding to the immobilizedreceptor. By implication, library members having two copies of the taghave two copies of the fusion protein containing the tag, and two copiesof a polypeptide to be screened. The library members that bind to thereceptor thus constitute the small subpopulation of library membersdisplaying two or more polypeptides. The library members not binding tothe receptor are the majority of library members which display fewerthan two copies of a polypeptide (i.e., zero or one copy). These librarymembers, which would nonspecifically bind to the immobilized target insubsequent steps without contributing any members capable of survivingaffinity screening through specific binding of the displayedpolypeptide, can thus be substantially eliminated.

The bound library members, which display multiple copies of polypeptide,can then be subject to one or more rounds of affinity screening to anyimmobilized target of interest. Because most library members that wouldotherwise contribute to nonspecific binding have been eliminated beforeaffinity screening to the target, each round of affinity screeningtypically results in a greater enrichment for library members withaffinity for the target than would be the case in conventional methods.The greater degree of enrichment per round of screening allows adequatescreening to be accomplished in fewer rounds and/or a greater proportionof the repertoire of specifically binding library members to beidentified.

So efficient are the selection methods of the invention that they resultin diverse populations in which the vast majority of members retainingfull-length coding sequences encode polypeptides having specificaffinity for the target. These polypeptides may differ in fine bindingspecificity within the target and binding affinity for the target.

A second aspect in which the invention represents a substantialdeparture from conventional methods resides in the subcloning of nucleicacids encoding displayed polypeptides of enriched libraries from adisplay vector to an expression vector without clonal isolation ofindividual members. The utility of transferring populations of codingsequences from a display vector to an expression vector without clonalisolation is realizable because the enriched libraries contain a highproportion of members having the desired binding specificity asdescribed above.

Subcloning is achieved by excising or amplifying nucleic acids encodingpolypeptides from the enriched library. The nucleic acids are thenpreferably size-fractionated on a gel and only full-length sequences areretained. The full-length sequences are inserted into an expressionvector in operable linkage to appropriate regulatory sequences to ensuretheir expression. The modified expression vector is then introduced intoappropriate host cells and expressed. Expression results in a populationof polypeptides having specific affinity for the desired target. Thepopulation of polypeptides can be purified from the host cells byconventional methods. The population of polypeptides typically hassubstantially the same members in substantially the same proportions aswere encoded by the enriched display library. As in the display library,the polypeptides typically differ in fine binding specificity, andbinding affinity for the chosen target.

The populations of polypeptides can be used as diagnostic andtherapeutic reagents. For example, if the target is a viral antigen, thepolypeptides can be used to assay the presence of the virus in tissuesamples. If the target is a tumor antigen, the polypeptides can be usedas a therapeutic reagent to deliver a toxic substance to cells bearingthe tumor antigen. The use of polyclonal preparation has advantages overa monoclonal reagent in both of these types of applications. Forexample, the diverse fine binding specificity of members of a populationoften allows the population to bind to several variant forms of target(e.g., species variants, escape mutant forms) to which a monoclonalreagent may be unable to bind.

II. Display Libraries

A. Replicable Genetic Packages

A replicable genetic package means a cell, spore or virus. Thereplicable genetic package can be eucaryotic or procaryotic. A displaylibrary is formed by introducing nucleic acids encoding exogenouspolypeptides to be displayed into the genome of the replicable geneticpackage to form a fusion protein with an endogenous protein that isnormally expressed from the outersurface of the replicable geneticpackage. Expression of the fusion protein, transport to the outersurfaceand assembly results in display of exogenous polypeptides from theoutersurface of the genetic package.

The genetic packages most frequently used for display libraries arebacteriophage, particularly filamentous phage, and especially phage M13,Fd and F1. Most work has inserted libraries encoding polypeptides to bedisplayed into either gIII or gVIII of these phage forming a fusionprotein. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989;MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO92/18619 (gene VIII). Such a fusion protein comprises a signal sequence,usually from a secreted protein other than the phage coat protein, apolypeptide to be displayed and either the gene III or gene VIII proteinor a fragment thereof. Exogenous coding sequences are often inserted ator near the N-terminus of gene III or gene VIII although other insertionsites are possible. Some filamentous phage vectors have been engineeredto produce a second copy of either gene III or gene VIII. In suchvectors, exogenous sequences are inserted into only one of the twocopies. Expression of the other copy effectively dilutes the proportionof fusion protein incorporated into phage particles and can beadvantageous in reducing selection against polypeptides deleterious tophage growth. In another variation, exogenous polypeptide sequences arecloned into phagemid vectors which encode a phage coat protein and phagepackaging sequences but which are not capable of replication. Phagemidsare transfected into cells and packaged by infection with helper phage.Use of phagemid system also has the effect of diluting fusion proteinsformed from coat protein and displayed polypeptide with wildtype copiesof coat protein expressed from the helper phage. See, e.g., Garrard, WO92/09690.

Eucaryotic viruses can be used to display polypeptides in an analogousmanner. For example, display of human heregulin fused to gp70 of Moloneymurine leukemia virus has been reported by Han et al., Proc. Natl. Acad.Sci. USA 92, 9747-9751 (1995). Spores can also be used as replicablegenetic packages. In this case, polypeptides are displayed from theoutersurface of the spore. For example, spores from B. subtilis havebeen reported to be suitable. Sequences of coat proteins of these sporesare provided by Donovan et al., J. Mol. Biol. 196, 1-10 (1987). Cellscan also be used as replicable genetic packages. Polypeptides to bedisplayed are inserted into a gene encoding a cell protein that isexpressed on the cells surface. Bacterial cells including Salmonellatyphimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae,Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis,Bacteroides nodosus, Moraxella bovis, and especially Escherichia coliare preferred. Details of outersurface proteins are discussed by Ladneret al., U.S. Pat. No. 5,571,698, and Georgiou et al., NatureBiotechnology 15, 29-34 (1997) and references cited therein. Forexample, the lamB protein of E. coli is suitable.

B. Displayed Polypeptides

Polypeptides typically displayed from replicable genetic packages fallinto a number of broad categories. One category is libraries of shortrandom or semi random peptides. See, e.g., Cwirla et al., supra. Thestrategy here is to produce libraries of short peptides in which some orall of the positions are systematically varied for the different aminoacids. Random peptide coding sequences can be formed by the cloning andexpression of randomly-generated mixtures of oligonucleotides ispossible in the appropriate recombinant vectors. See, e.g., Oliphant etal., Gene 44;177-183 (1986) A second category of library comprisesvariants of a starting framework protein. See Ladner et al., supra. Inthis approach, a starting polypeptide which may be of substantial lengthis chosen and only selected positions are varied. The nucleic acidencoding the starting polypeptide can be mutagenized by, for example,insertion of mutagenic cassette(s) or error-prone PCR.

A third category of library consists of antibody libraries. Antibodylibraries can be single or double chain. Single chain antibody librariescan comprise the heavy or light chain of an antibody alone or thevariable domain thereof. However, more typically, the members ofsingle-chain antibody libraries are formed from a fusion of heavy andlight chain variable domains separated by a peptide spacer within asingle contiguous protein. See e.g., Ladner et al., WO 88/06630;McCafferty et al., WO 92/01047. Double-chain antibodies are formed bynoncovalent association of heavy and light chains or binding fragmentsthereof. The diversity of antibody libraries can arise from obtainingantibody-encoding sequences from a natural source, such as a nonclonalpopulation of immunized or unimmunized B cells. Alternatively, oradditionally, diversity can be introduced by artificial mutagenesis asdiscussed for other proteins.

Nucleic acids encoding polypeptides to be displayed optionally flankedby spacers are inserted into the genome of a replicable genetic packageas discussed above by standard recombinant DNA techniques (seegenerally, Sambrooke et al., Molecular Cloning, A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, incorporated by reference herein). The nucleic acids areultimately expressed as polypeptides (with or without spacer orframework residues) fused to all or part of the an outersurface proteinof the replicable package. Libraries often have sizes of about 10³, 10⁴,10⁶, 10⁷, 10⁸ or more members.

C. Double-Chain Antibody Display Libraries

Double-chain antibody display libraries represent a species of thereplicable genetic display libraries discussed above. Production of suchlibraries is described by, e.g., Dower, U.S. Pat. No. 5,427,908; Huse WO92/06204; Huse, in Antibody Engineering, (Freeman 1992), Ch. 5; Kang, WO92/18619; Winter, WO 92/20791; McCafferty, WO 92/01047; Hoogenboom WO93/06213; Winter et al., Annu. Rev. Immunol. 12, 433-455 (1994);Hoogenboom et al., Immunological Reviews 130, 41-68 (1992); Soderlind etal., Immunological Reviews 130, 109-124 (1992). In double-chain antibodylibraries, one antibody chain is fused to a phage coat protein, as isthe case in single chain libraries. The partner antibody chain iscomplexed with the first antibody chain, but the partner is not directlylinked to a phage coat protein. Either the heavy or light chain can bethe chain fused to the coat protein. Whichever chain is not fused to thecoat protein is the partner chain. This arrangement is typicallyachieved by incorporating nucleic acid segments encoding one antibodychain gene into either gIII or gVIII of a phage display vector to form afusion protein comprising a signal sequence, an antibody chain, and aphage coat protein. Nucleic acid segments encoding the partner antibodychain can be inserted into the same vector as those encoding the firstantibody chain. Optionally, heavy and light chains can be inserted intothe same display vector linked to the same promoter and transcribed as apolycistronic message. Alternatively, nucleic acids encoding the partnerantibody chain can be inserted into a separate vector (which may or maynot be a phage vector). In this case, the two vectors are expressed inthe same cell (see WO 92/20791). The sequences encoding the partnerchain are inserted such that the partner chain is linked to a signalsequence, but is not fused to a phage coat protein. Both antibody chainsare expressed and exported to the periplasm of the cell where theyassemble and are incorporated into phage particles.

Antibody encoding sequences can be obtained from lymphatic cells of ahuman or nonhuman animal. Often the cells have been immunized, in whichcase immunization can be performed in vivo before harvesting cells, orin vitro after harvesting cells, or both. Spleen cells of an immunizedanimal are a preferred source material. Immunization of humans is onlypossible with certain antigens. The number of different H chain genesand L chain genes in a spleen from an immunized animal is about 10⁶,which can be assembled in 10¹² potential combinations.

Rearranged immunoglobulin genes can be cloned from genomic DNA or mRNA.For the latter, mRNA is extracted from the cells and cDNA is preparedusing reverse transcriptase and poly dT oligonucleotide primers. Primersfor cloning antibody encoding sequences are discussed by Larick et al.,Bio/Technology 7, 934 (1989), Danielsson & Borrebaceick, in AntibodyEngineering: A Practical Guide (Freeman, NY, 1992), p. 89 and Huse, id.at Ch. 5.

Repertoires of antibody fragments have been constructed by combiningamplified VH and VL sequences together in several ways. Light and heavychains can be inserted into different vectors and the vectors combinedin vitro (Hogrefe et al., Gene 128, 119-126 (1993)) or in vivo(Waterhouse et al., Nucl. Acids. Res. 21, 2265-66 (1993)).Alternatively, the light and heavy chains can be cloned sequentiallyinto the same vector (Barbas et al., Proc. Natl. Acad. Sci. USA 88,7987-82 (1991)) or assembled together by PCR and then inserted into avector (Clackson et al., Nature 352, 624-28 (1991)). Repertoires ofheavy chains can be also be combined with a single light chain or viceversa. Hoogenboom et al., J. Mol. Biol. 227, 381-88 (1992).

Some exemplary vectors and procedures for cloning populations of heavychain and light chain encoding sequences have been described by Huse, WO92/06204. Diverse populations of sequences encoding Hc polypeptides arecloned into M13IX30 and sequences encoding Lc polypeptides are clonedinto M13IX11. The populations are inserted between the XhoI-SeeI or StuIrestriction enzyme sites in M13IX30 and between the SacI-XbaI or EcoRVsites in M13IX11 (FIGS. 1A and B of Huse, respectively). Both vectorscontain two pairs of MluI-HindIII restriction enzyme sites (FIGS. 1A andB of Huse) for joining together the Hc and Lc encoding sequences andtheir associated vector sequences. The two pairs are symmetricallyorientated about the cloning site so that only the vector proteinscontaining the sequences to be expressed are exactly combined into asingle vector.

Others exemplary vectors and procedures for cloning antibody chains intofilamentous phage are described in the present Examples.

III. Enrichment for Polyvalent Display Members

A. Theory of the method

That members of a library displaying multiple copies of a polypeptideare comparatively rare in display libraries is a finding apparently atvariance with some early reports in the field. See, e.g., Cwirla et al.,supra. Most work on display libraries has been done by inserting nucleicacid libraries into pIII or pVIII of filamentous phage. Because pIII ispresent in 4 or 5 copies per phage and pVIII is present in severalhundred copies per phage, some early reports assumed that foreignpolypeptides would be displayed in corresponding numbers per phage.However, more recent work has made clear that the actual number ofcopies of polypeptide displayed per phage is well below theoreticalexpectations, perhaps due to proteolytic cleavage of polypeptides.Winter et al., Ann. Rev. Immunol. 12, 433-55 (1994). Further, vectorsystems used for phage display often encode two copies of a phage coatprotein, one of which is a wildtype protein and the other of which formsa fusion protein with exogenous polypeptides to be displayed. Bothcopies are expressed and the wildtype coat protein effectively dilutesthe representation of the fusion protein in the phage coat.

A typical ratio of displayed Fabs per phage, when Fabs are expressedfrom pVIII of a filamentous phage is about 0.2. The probability, Pr(y),of y Fabs being expressed on a phage particle if the average frequencyof expression per phage is n is given by the Poisson probabilitydistribution

Pr(y)=e ^(−n) n ^(y) /y!

For a frequency of 0.2 Fabs per phage, the probabilities for theexpression of 0, 1, 2, and 3 Fabs per phage are 0.82, 0.16, 0.016, and0.0011. The proportion of phage particle displaying two or more Fabs istherefore only 0.017.

The low representation of members displaying more than one Fab fragmentin a phage display library can be related to the present inventors'result that only a small percentage of such library members are capableof surviving affinity selection to immobilized binding partners. Alibrary was constructed in which all members encoded the same Fabfragment which was known to have a high binding affinity for aparticular target. It was found that even under the mildest separationconditions for removal of free from bound phage, it was not possible tobind more than about 0.004 of the total phage. This proportion is thesame order of magnitude as the proportion of phage displaying at leasttwo Fab fragments, suggesting that phage must display at least two Fabfragments to bind to immobilized target. Probably shear forcesdissociate phage displaying only a single Fab fragment from the solidphase. Therefore, at least two binding events are necessary for aphage-Fab library member to be bound to immobilized target withsufficient avidity to enable separation of the bound from the freephage. It is expected that similar constraints apply in other forms ofdisplay library.

The strategy of the present invention is to enrich for library membersdisplaying more than one polypeptide before the library is contactedwith a screening target. Library members lacking two or morepolypeptides, which are incapable of surviving affinity via bindingthrough displayed polypeptides to any immobilized screening target, butwhich nevertheless can survive affinity selection by formation ofmultiple nonspecific bonds to such a target or its support, are thussubstantially eliminated before screening of the library to target isperformed.

B. Tags and Receptors

The above strategy is effected by the use of paired tags and receptors.A tag is typically a short peptide sequence and a receptor is any agentthat shows specific but reversible binding for the tag and can beimmobilized to a support. Suitable tag-receptor combinations includeepitope and antibody; for example, many high affinity hexapeptideligands are known for the anti-dynorphin mAb 32.39, (see Barrett et al.,Neuropeptides 6, 113-120 (1985) and Cull et al., PNAS 89, 1865-1869(1992)) and a variety of short peptides are known to bind the MAb 3E7(Schatz, Biotechnology 11, 1138-43 (1993)). Another combination of tagand antibody is described by Blanar & Rutter, Science 256, 1014-1018(1992).

Another example of a tag-receptor pair is the FLAG™ system (Kodak). TheFLAG™ molecular tag consists of an eight amino acid FLAG peptide markerthat is linked to the target binding moiety. A 24 base pair segmentcontaining a FLAG coding sequence can be inserted adjacent to anucleotide sequence that codes for the displayed polypeptide. The FLAGpeptide includes an enterokinase recognition site that corresponds tothe carboxy-terminal five amino acids. Capture moieties suitable for usewith the FLAG peptide marker include antibodies Anti-FLAG M1, M2 and M5,which are commercially available.

Still other combinations of peptides and antibodies can be identified byconventional phage display methods. Further suitable combinations ofpeptide sequence and receptor include polyhistidine and metal chelateligands containing Ni²⁺ immobilized on agarose (see Hochuli in GeneticEngineering: Principles and Methods (ed. J K Setlow, Plenum Press, NY),Ch. 18, pp. 87-96 and maltose binding protein (Maina et al., Gene 74,365-373 (1988)).

Receptors are often labelled with biotin allowing the receptors to beimmobilized to an avidin-coated support. Biotin labelling can beperformed using the biotinylating enzyme, BirA (see, e.g., Schatz,Biotechnology 11, 1138-43 (1993)).

A nucleic acid sequence encoding a tag is inserted a into a displayvector in such a manner that the tag is expressed as part of the fusionprotein containing the polypeptide to be displayed and an outersurfaceprotein of the replicable genetic package. The relative ordering ofthese components is not critical provided that the tag and polypeptideto be displayed are both exposed on the outersurface of the package. Forexample, the tag can be placed between the outersurface protein and thedisplayed polypeptide or at or near the exposed end of the fusionprotein.

In replicable genetic packages displaying Fabs, a tag can be fused toeither the heavy or the light Fab chain, irrespective which chain islinked to a phage coat protein. Optionally, two different tags can usedone fused to each of the heavy and light chains. One tag is usuallypositioned between the phage coat protein and antibody chain linkedthereto, and the other tag is positioned at either the N- or C-terminusof the partner chain.

C. Selection of Polyvalent Members

Selection of polyvalent library members is performed by contacting thelibrary with the receptor for the tag component of library members.Usually, the library is contacted with the receptor immobilized to asolid phase and binding of library members through their tag to thereceptor is allowed to reach equilibrium. The complexed receptor andlibrary members are then brought out of solution by addition of a solidphase to which the receptor bears affinity (e.g., an avidin-labelledsolid phase can be used to immobilize biotin-labelled receptors).Alternatively, the library can be contacted with receptor in solutionand the receptor subsequently immobilized. The concentration of receptorshould usually be at or above the Kd of the tag/receptor during solutionphase binding so that most displayed tags bind to a receptor atequilibrium. When the receptor-library members are contacted with thesolid phase only the library members linked to receptor through at leasttwo displayed tags remain bound to the solid phase following separationof the solid phase from library members in solution. Library memberslinked to receptor through a single tag are presumably sheared from thesolid phase during separation and washing of the solid phase. Afterremoval of unbound library members, bound library members can bedissociated from the receptor and solid phase by a change in ionicstrength or pH, or addition of a substance that competes with the tagfor binding to the receptor. For example, binding of metal chelateligands immobilized on agarose and containing Ni²⁺ to a hexahistidinesequence is easily reversed by adding imidazole to the solution tocompete for binding of the metal chelate ligand. Antibody-peptidebinding can often be dissociated by raising the pH to 10.5 or higher.

The average number of polypeptides per library member selected by thismethod is affected by a number of factors. Decreasing the concentrationof receptor during solution-phase binding has the effect of increasingthe average number of polypeptides in selected library members. Anincrease in the stringency of the washing conditions also increases theaverage number of polypeptides per selected library member. The physicalrelationship between library members and the solid phase can also bemanipulated to increase the average number of polypeptides per librarymember. For example, if discrete particles are used as the solid phase,decreasing the size of the particles increases the steric constraints ofbinding and should require a higher density of polypeptides displayedper library member.

For Fab libraries having two tags, one linked to each antibody chain,two similar rounds of selection can be performed, with the products ofone round becoming the starting materials for the second round. Thefirst round of selection is performed with a receptor to the first tag,and the second round with a receptor to the second tag. Selecting forboth tags enriches for library members displaying two copies of bothheavy and light antibody chains (i.e., two Fab fragments).

D. Selection For Affinity to Target

Library members enriched for polyvalent display of Fabs or otherpolypeptides are screened for binding to a target. The target can be anymolecule of interest for which it is desired to identify bindingpartners. The target should lack specific binding affinity for thetag(s), because in this step it is the displayed polypeptides beingscreened, and not the tags that bind to the target. The screeningprocedure at this step is closely analogous to that in the previous stepexcept that the affinity reagent is a target of interest rather than areceptor to a tag. The enriched library members are contacted with thetarget which is usually labelled (e.g., with biotin) in such a mannerthat allows its immobilization. Binding is allowed to proceed toequilibrium and then target is brought out of solution by contactingwith the solid phase in a process known as panning (Parmley & Smith,Gene 73, 305-318 (1988)). Library members that remain bound to the solidphase throughout the selection process do so by virtue of polyvalentbonds between them and immobilized target molecules. Unbound librarymembers are washed away from the solid phase.

Usually, library members are subject to amplification before performinga subsequent round of screening. Often, bound library members can beamplified without dissociating them from the support. For example, geneVIII phage library members immobilized to beads, can be amplified byimmersing the beads in a culture of E. coli. Likewise, bacterial displaylibraries can be amplified by adding growth media to bound librarymembers. Alternatively, bound library members can be dissociated fromthe solid phase (e.g., by change of ionic strength or pH) beforeperforming subsequent selection, amplification or propagation.

After affinity selection, bound library members are now enriched for twofeatures: multivalent display of polypeptides and display ofpolypeptides having specific affinity for the target of interest.However, after subsequent amplification, to produce a secondary library,the secondary library remains enriched for display of polypeptideshaving specific affinity for the target, but, as a result ofamplification, is no longer enriched for polyvalent display ofpolypeptides. Thus, a second cycle of polyvalent enrichment can then beperformed, followed by a second cycle of affinity enrichment to thescreening target. Further cycles of affinity enrichment to the screeningtarget, optionally, alternating with amplification and enrichment forpolyvalent display can then be performed, until a desired degree ofenrichment has been performed.

In a variation, affinity screening to a target is performed incompetition with a compound that resembles but is not identical to thetarget. Such screening preferentially selects for library members thatbind to a target epitope not present on the compound. In a furthervariation, bound library members can be dissociated from the solid phasein competition with a compound having known crossreactivity with atarget for an antigen. Library members having the same or similarbinding specificity as the known compound relative to the target arepreferentially eluted. Library members with affinity for the targetthrough an epitope distinct from that recognized by the compound remainbound to the solid phase.

Discrimination in selecting between polypeptides of different monovalentaffinities for the target is affected by the valency of library membersand the concentration of target during the solution phase binding.Assuming a minimum of i labeled target molecules must be bound to alibrary member to immobilize it on a solid phase, then the probabilityof immobilization can be calculated for a library member displaying npolypeptides. From the law of mass action, the bound/free polypeptidefraction and the bound/polypeptide fraction, F, is K[targ]/(1+K[targ]),where [targ] is the total target concentration in solution. Thus, theprobability that i or more displayed polypeptides per library member arebound by the labeled target ligand is given by the binomial probabilitydistribution:$\sum\limits_{y = i}^{n}\left( {{{n!}/\left\lbrack {{y!}{\left( {n - y} \right)!}} \right\rbrack}{F^{y}\left( {1 - F} \right)}^{n - y}} \right.$

As the probability is a function of K and [target], multivalent displaymembers each having a monovalent affinity, K, for the target can beselected by varying the concentration of target. The probabilities ofsolid-phase immobilization for i=1, 2, or 3, with library membersexhibiting monovalent affinities of 0.1/[Ag], 1/[Ag], and 10/[Ag], anddisplaying n polypeptides per member are:

n K = 0.1/[targ] K = 1/[targ] K = 10/[targ] Probability ofImmobilization (i = 1) 1 0.09 0.5 0.91 2 0.17 0.75 0.99 3 0.25 0.875 40.32 0.94 5 0.38 0.97 6 0.44 0.98 7 0.49 0.99 8 0.53 9 0.58 10 0.61 200.85 50 0.99 Probability of Immobilization (i = 2) 2 0.008 0.25 0.83 30.023 0.50 0.977 4 0.043 0.69 0.997 5 0.069 0.81 6 0.097 0.89 7 0.1280.94 8 0.160 0.965 9 0.194 0.98 20 0.55 50 0.95 Probability ofImmobilization (i = 3) 3 0.00075 0.125 0.75 4 0.0028 0.31 0.96 5 0.00650.50 0.99 6 0.012 0.66 7 0.02 0.77 8 0.03 0.855 9 0.0415 0.91 10 0.0550.945 12 0.089 0.98 14 0.128 0.99 20 0.27 50 0.84

The above tables show that the discrimination between immobilizingpolypeptides of different monovalent binding affinities is affected bythe valency of library members (n) and by the concentration of targetfor the solution binding phase. Discrimination is maximized when n(number of polypeptides displayed per phage) is equal to i (minimumvalency required for solid phase binding). Discrimination is alsoincreased by lowering the concentration of target during the solutionphase binding. Usually, the target concentration is around the Kd of thepolypeptides sought to be isolated. Target concentration of 10⁻⁸-10⁻¹⁰ Mare typical.

Enriched libraries produced by the above methods are characterized by ahigh proportion of members encoding polypeptides having specificaffinity for the target. For example, at least 10, 25, 50, 75, 95, or99% of members encode polypeptides having specific affinity for thetarget. The exact percentage of members having affinity for the targetdepends whether the library has been amplified following selection,because amplification increases the representation of genetic deletions.However, among members with full-length polypeptide coding sequences,the proportion encoding polypeptides with specific affinity for thetarget is very high (e.g., at least 50, 75, 95 or 99%). Not all of thelibrary members that encode a polypeptide with specific affinity for thetarget necessarily display the polypeptide. For example, in a library inwhich 95% of members with full-length coding sequences encodepolypeptides with specific affinity for the target, usually fewer thanhalf actually display the polypeptide. Usually, such libraries have atleast 4, 10, 20, 50, 100, 1000, 10,000 or 100,000 different codingsequences. Usually, the representation of any one such coding sequencesis no more than 50%, 25% or 10% of the total coding sequences in thelibrary.

IV. Polyclonal Libraries

A. Production

The nucleic acid sequences encoding displayed polypeptides such as areproduced by the above methods can be subcloned directly into anexpression vector without clonal isolation and testing of individualmembers. Generally, the sequence encoding the outersurface protein ofthe display vector fused to displayed polypeptides is not excised oramplified in this process. The nucleic acids can be excised byrestriction digestion of flanking sequences or can be amplified by PCRusing primers to sites flanking the coding sequences. See generally PCRTechnology: Principles and Applications for DNA Amplification (ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19:4967 (1991); Eckertet al., PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson etal., IRL Press, Oxford). PCR primers can contain a marker sequence thatallows positive selection of amplified fragments when introduced into anexpression vector. PCR primers can also contain restriction sites toallow cloning into an expression vector, although this is not necessary.For Fab libraries, if heavy and light chains are inserted adjacent orproximate to each other in a display vector, the two chains can beamplified or excised together. For some Fab libraries, only the variabledomains of antibody chain(s) are excised or amplified. If the heavy orlight chains of a Fab library are excised or amplified separately, theycan subsequently be inserted into the same or different expressionvectors.

Having excised or amplified fragments encoding displayed polypeptides,the fragments are usually size-purified on an agarose gel or sucrosegradient. Typically, the fragments run as a single sharp full-lengthband with a smear at lower molecular corresponding to various deletedforms of coding sequence. The band corresponding to full-length codingsequences is removed from the gel or gradient and these sequences areused in subsequent steps.

The next step is to join the nucleic acids encoding full-length codingsequences to an expression vector thereby creating a population ofmodified forms of the expression vector bearing different inserts. Thiscan be done by conventional ligation of cleaved expression vector withinserts cleaved to have compatible ends. Alternatively, the use ofrestriction enzymes on insert DNA can be avoided. This method of cloningis beneficial because naturally encoded restriction enzyme sites may bepresent within insert sequences, thus, causing destruction of thesequence when treated with a restriction enzyme. For cloning withoutrestricting, the insert and linearized vector sequences are treatedbriefly with a 3′ to 5′ exonuclease such as T4 DNA polymerase orexonuclease III. See Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd Ed., CSHP, New York 1989). The protruding 5′ termini of theinsert generated by digestion are complementary to single-strandedoverhangs generated by digestion of the vector. The overhangs areannealed, and the reannealed vector transfected into recipient hostcells. The same result can be accomplished using 5′ to 3′ exonucleasesrather than a 3′ to 5′ exonuclease.

Preferably, ligation of inserts to expression vector is performed underconditions that allow selection against reannealed vector and uncutvector. A number of vectors containing conditional lethal genes thatallow selection against reannealed vector under nonpermissive conditionsare known. See, e.g., Conley & Saunders, Mol. Gen. Genet. 194, 211-218(1984). These vectors effectively allow positive selection for vectorshaving received inserts. Selection can also be accomplished by cleavingan expression vector in such a way that a portion of a positiveselection marker (e.g., antibiotic resistance) is deleted. The missingportion is then supplied by full-length inserts. The portion can beintroduced at the 3′ end of polypeptide coding sequences in the displayvector, or by included in a primer used for amplification of the insert.An exemplary selection scheme, in which inserts supply a portion of atetracycline-resistance gene promoter deleted by HindIII cleavage of apBR-derivative vector, is described in Example 17.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the vector includes apromoter and other regulatory sequences in operable linkage to theinserted coding sequences that ensure the expression of the latter. Useof an inducible promoter is advantageous to prevent expression ofinserted sequences except under inducing conditions. Inducible promotersinclude arabinose, lacZ, metallothionein promoter or a heat shockpromoter. Cultures of transformed organisms can be expanded undernoninducing conditions without biasing the population for codingsequences whose expression products are better tolerated by the hostcells. The vector may also provide a secretion signal sequence positionto form a fusion protein with polypeptides encoded by insertedsequences, although often inserted polypeptides are linked to a signalsequences before inclusion in the vector. Vectors to be used to receivesequences encoding antibody light and heavy chain variable domainssometimes encode constant regions or parts thereof that can be expressedas fusion proteins with inserted chains thereby leading to production ofintact antibodies or fragments thereof.

E. coli is one prokaryotic host useful particularly for cloning thepolynucleotides of the present invention. Other microbial hosts suitablefor use include bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

Mammalian tissue cell culture can also be used to express and producethe polypeptides of the present invention (see Winnacker, From Genes toClones (VCH Publishers, N.Y., N.Y., 1987). A number of suitable hostcell lines capable of secreting intact immunoglobulins have beendeveloped including the CHO cell lines, various Cos cell lines, HeLacells, myeloma cell lines, transformed B-cells and hybridomas.Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, and an enhancer(Queen et al., Immunol. Rev. 89:49-68 (1986)), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus, orcytomegalovirus.

Methods for introducing vectors containing the polynucleotide sequencesof interest vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment or electroporation may beused for other cellular hosts. (See generally Sambrook et al., supra).

Once expressed collections of antibodies or other polypeptides arepurified from culture media and host cells. Usually, polypeptides areexpressed with signal sequences and are thus released to the culturemedia. However, if polypeptides are not naturally secreted by hostcells, the polypeptides can be released by treatment with milddetergent. Polypeptides can then be purified by conventional methodsincluding ammonium sulfate precipitation, affinity chromatography toimmobilized target, column chromatography, gel electrophoresis and thelike (see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,1982)).

B. Characteristics of Libraries

The above methods result in novel libraries of nucleic acid sequencesencoding polypeptides having specific affinity for a chosen target. Thelibraries of nucleic acids typically have at least 5, 10, 20, 50, 100,1000, 10⁴ or 10⁵ different members. Usually, no single memberconstitutes more than 25 or 50% of the total sequences in the library.Typically, at least 75, 90, 95, 99 or 99.9% of library members encodepolypeptides with specific affinity for the target molecules. Thenucleic acid libraries can exist in free form, as components of anyvector or transfected as a component of a vector into host cells.

The nucleic acid libraries can be expressed to generate polyclonallibraries of antibodies or other polypeptides having specific affinityfor a target. The composition of such libraries is determined from thecomposition of the nucleotide libraries. Thus, such libraries typicallyhave at least 5, 10, 20, 50, 100, 1000, 10⁴ or 10⁵ members withdifferent amino acid composition. Usually, no single member constitutesmore than 25 or 50% of the total polypeptides in the library. In somelibraries, at least 75, 90, 95, 99 or 99.9% of polypeptides havespecific affinity for the target molecules. The different polypeptidesdiffer from each other in terms of fine binding specificity and affinityfor the target.

V. Diagnostic and Therapeutics Uses

The use of polyclonal antibodies in diagnostics and therapeutics hasbeen limited by the inability to generate preparations that have awell-defined affinity and specificity. Monoclonal antibodies developedusing hybridoma technology do have well-defined specificity andaffinity, but the selection process is often long and tedious. Further,a single monoclonal antibody does not meet all of the desiredspecificity requirements. Formation of polyclonal mixtures by isolation,and characterization of individual monoclonal antibodies, which are thenmixed would be time consuming process which would increase in proportionto the number of monoclonal, included in the mixture and becomeprohibitive for substantial numbers of monoclonals. The polyclonallibraries of antibodies and other polypeptides having specificity for agiven target produced by the present methods avoid these difficulties,and provide reagents that are useful in many therapeutic and diagnosticapplications.

The use of polyclonals has a number of advantages with respect tomonoclonals. By binding to multiple sites on a target, polyclonalantibodies or other polypeptides can generate a stronger signal (fordiagnostics) or greater blocking/inhibition/cytotoxicity (fortherapeutics) than a monoclonal that binds to a single site. Further, apolyclonal preparation can bind to numerous variants of a prototypicaltarget sequence (e.g., allelic variants, species variants, strainvariants, drug-induced escape variants) whereas a monoclonal antibodymay bind only to the prototypical sequence or a narrower range ofvariants thereto.

Polyclonal preparations of antibodies and other polypeptides can beincorporated into compositions for diagnostic or therapeutic use. Thepreferred form depends on the intended mode of administration anddiagnostic or therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like. See Remington's Pharmaceutical Science, (15thed., Mack Publishing Company, Easton, Pa., 1980). Compositions intendedfor in vivo use are usually sterile.

Although the invention has been described in detail for purposes ofclarity of understanding, it will be obvious that certain modificationsmay be practiced within the scope of the appended claims. Allpublications and patent documents cited in this application are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted. Cell lines producingantibodies designated 7F11, CD43.5.PC, CD43.9, CDTXA1.PC, referred to inthe following examples, have been deposited with the American TypeCulture Collection, 10801 Uniiversity Boulevard, Manassas, Va.20110-2209 on Apr. 3 and Dec. 5, 1997 under the Budapest Treaty andgiven the Accession Nos. HB12443, 98389, 98390, and 98388, respectively.These deposit will be maintained at an authorized depository andreplaced in the event of mutation, nonviability or destruction for aperiod of at least five years after the most recent request for releaseof a sample was received by the depository, for a period of at leastthirty years after the date of the deposit, or during the enforceablelife of the related patent, whichever period is longest. Allrestrictions on the availability to the public of these cell lines willbe irrevocably removed upon the issuance of a patent from theapplication.

EXAMPLES Example 1 Immunization of Mice With Antigens and Purificationof RNA From Mouse Spleens

Mice were immunized by the following method based on experience of thetiming of spleen harvest for optimal recovery of mRNA coding forantibody. Two species of mice were used: Balb/c (Charles RiverLaboratories, Wilmington, Mass.) and A/J (Jackson Laboratories, BarHarbor, Me.). Each of ten mice were immunized intraperitoneally withantigen using 50 μg protein in Freund's complete adjuvant on day 0, andday 28. Tests bleeds of mice were obtained through puncture of theretro-orbital sinus. If, by testing the titers, they were deemed high byELISA using biotinylated antigen immobilized via streptavidin, the micewere boosted with 50 μg of protein on day 70, 71 and 72, with subsequentsacrifice and splenectomy on day 77. If titers of antibody were notdeemed satisfactory, mice were boosted with 50 μg antigen on day 56 anda test bleed taken on day 63. If satisfactory titers were obtained, theanimals were boosted with 50 μg of antigen on day 98, 99, and 100 andthe spleens harvested on day 105. Typically, a test bleed dilution of1:3200 or more resulted in a half maximal ELISA response to beconsidered satisfactory.

The spleens were harvested in a laminar flow hood and transferred to apetri dish, trimming off and discarding fat and connective tissue. Thespleen was, working quickly, macerated with the plunger from a sterile 5cc syringe in the presence of 1.0 ml of solution D (25.0 g guanidinethiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3 ml sterilewater, 1.76 ml 0.75 M sodium citrate (pH 7.0), 2.64 ml 10% sarkosyl(Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (FisherScientific, Pittsburgh, Pa.)). The spleen suspension was pulled throughan 18 gauge needle until viscous and all cells were lysed, thentransferred to a microcentrifuge tube. The petri dish was washed with100 μl of solution D to recover any remaining spleen, and this wastransferred to the tube. The suspension was then pulled through a 22gauge needle an additional 5-10 times. The sample was divided evenlybetween two microcentrifuge tubes and the following added in order, withmixing by inversion after each addition: 100 μl 2 M sodium acetate (pH4.0), 1.0 ml water-saturated phenol (Fisher Scientific, Pittsburgh,Pa.), 200 μl chloroform/isoamyl alcohol 49:1 (Fisher Scientific,Pittsburgh, Pa.). The solution was vortexed for 10 seconds and incubatedon ice for 15 min. Following centrifugation at 14 krpm for 20 min at2-8° C., the aqueous phase was transferred to a fresh tube. An equalvolume of water saturated phenol/chloroform/isoamyl alcohol (50:49:1)was added, and the tube was vortexed for ten seconds. After a 15 minincubation on ice, the sample was centrifuged for 20 min at 2-8° C., andthe aqueous phase was transferred to a fresh tube and precipitated withan equal volume of isopropanol at −20° C. for a minimum of 30 min.Following centrifugation at 14 krpm for 20 min at 4° C., the supernatantwas aspirated away, the tubes briefly spun and all traces of liquidremoved. The RNA pellets were each dissolved in 300 μl of solution D,combined, and precipitated with an equal volume of isopropanol at −20°C. for a minimum of 30 min. The sample was centrifuged 14 krpm for 20min at 4° C., the supernatant aspirated as before, and the sample rinsedwith 100 μl of ice-cold 70% ethanol. The sample was again centrifuged 14krpm for 20 min at 4° C., the 70% ethanol solution aspirated, and theRNA pellet dried in vacuo. The pellet was resuspended in 100 μl ofsterile distilled water. The concentration was determined by A₂₆₀ usingan absorbance of 1.0 for a concentration of 40 μg/ml. The RNA was storedat −80° C.

Example 2 Preparation of Complementary DNA (cDNA)

The total RNA purified as described above was used directly as templatefor cDNA. RNA (50 μg) was diluted to 100 μL with sterile water, and 10μL-130 ng/μL oligo dT₁₂ (synthesized on Applied Biosystems Model 392 DNAsynthesizer at Biosite Diagnostics) was added. The sample was heated for10 min at 70° C., then cooled on ice. 40 μL 5×first strand buffer wasadded (Gibco/BRL, Gaithersburg, Md.), 20/μL 0.1 M dithiothreitol(Gibco/BRL, Gaithersburg, Md.), 10 μL 20 mM deoxynucleosidetriphosphates (dNTP's, Boehringer Mannheim, Indianapolis, Ind.), and 10μL water on ice. The sample was then incubated at 37° C. for 2 min. 10μL SUPERSCRIPT™ II.reverse transcriptase (Gibco/BRL, Gaithersburg, Md.)was added and incubation was continued at 37° C. for 1 hr. The cDNAproducts were used directly for polymerase chain reaction (PCR).

Example 3 Amplification of cDNA by PCR

To amplify substantially all of the H and L chain genes using PCR,primers were chosen that corresponded to substantially all publishedsequences. Because the nucleotide sequences of the amino terminals of Hand L contain considerable diversity, 33 oligonucleotides weresynthesized to serve as 5′ primers for the H chains (FIG. 1), and 29oligonucleotides were synthesized to serve as 5′ primers for the kappa Lchains (FIG. 2). The 5′ primers were made according to the followingcriteria. First, the second and fourth amino acids of the L chain andthe second amino acid of the heavy chain were conserved. Mismatches thatchanged the amino acid sequence of the antibody were allowed in anyother position. Second, a 20 nucleotide sequence complementary to theM13 uracil template was synthesized on the 5′ side of each primer. Thissequence is different between the H and L chain primers, correspondingto 20 nucleotides on the 3′ side of the pelB signal sequence for L chainprimers and the alkaline phosphatase signal sequence for H chainprimers. The constant region nucleotide sequences required only one 3′primer each to the H chains and the kappa L chains (FIG. 2).Amplification by PCR was performed separately for each pair of 5′ and 3′primers. A 50 μL reaction was performed for each primer pair with 50pmol of 5′ primer, 50 pmol of 3′ primer, 0.25 μL Taq DNA Polymerase (5units/μL Boehringer Mannheim, Indianapolis, Ind.), 3 μL cDNA (describedin Example 2), 5 μL 2 mM dNTP's, 5 μL 10×Taq DNA polymerase buffer withMgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to 50 μL.Amplification was done using a GENEAMP® 9600 thermal cycler (PerkinElmer, Foster City, Calif.) with the following program: 94° C. for 1min; 30 cycles of 94° C. for 20 sec. 55° C. for 30 sec, and 72° C. for30 sec; 72° C. for 6 min; 4° C.

The dsDNA products of the PCR process were then subjected to asymmetricPCR using only 3′ primer to generate substantially only the anti-sensestrand of the target genes. A 100 μL reaction was done for each dsDNAproduct with 200 pmol of 3′ primer, 2 μL of ds-DNA product, 0.5 μL TaqDNA Polymerase, 10 μL 2 mM dNTP's, 10 μL 10×Taq DNA polymerase bufferwith MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to 100 μL.The same PCR program as that described above was used to amplify thesingle-stranded (ss)-DNA.

Example 4 Purification of ss-DNA by High Performance LiquidChromatography and Kinasing ss-DNA

The H chain ss-PCR products and the L chain ss-PCR products were ethanolprecipitated by adding 2.5 volumes ethanol and 0.2 volumes 7.5 Mammonium acetate and incubating at −20° C. for at least 30 min. The DNAwas pelleted by centrifuging in an Eppendorf centrifuge at 14 krpm for10 min at 2-8° C. The supernatant was carefully aspirated, and the tubeswere briefly spun a 2nd time. The last drop of supernatant was removedwith a pipet. The DNA was dried in vacuo for 10 min on medium heat. TheH chain products were pooled in 210 μL water and the L chain productswere pooled separately in 210 μL water. The ss-DNA was purified by highperformance liquid chromatography (HPLC) using a Hewlett Packard 1090HPLC and a GENPAK™ FAX anion exchange column (Millipore Corp., Milford,Mass.). The gradient used to purify the ss-DNA is shown in Table 1, andthe oven temperature was at 60° C. Absorbance was monitored at 260 nm.The ss-DNA eluted from the HPLC was collected in 0.5 min fractions.Fractions containing ss-DNA were ethanol precipitated, pelleted anddried as described above. The dried DNA pellets were pooled in 200 μLsterile water.

TABLE 1 HPLC gradient for purification of ss-DNA Time (min) % A % B % CFlow (mL/min) 0 70 30 0 0.75 2 40 60 0 0.75 32 15 85 0 0.75 35 0 100 00.75 40 0 100 0 0.75 41 0 0 100 0.75 45 0 0 100 0.75 46 0 100 0 0.75 510 100 0 0.75 52 70 30 0 0.75 Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0Buffer B is 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 8.0 Buffer C is 40 mmphosphoric acid

The ss-DNA was kinased on the 5′ end in preparation for mutagenesis(Example 7). 24 μL 10×kinase buffer (United States Biochemical,Cleveland, Ohio), 10.4 μL 10 mM adenosine-5′-triphosphate (BoehringerMannheim, Indianapolis, Ind.), and 2 μL polynucleotide kinase (30units/μL, United States Biochemical, Cleveland, Ohio) was added to eachsample, and the tubes were incubated at 37° C. for 1 hr. The reactionswere stopped by incubating the tubes at 70° C. for 10 min. The DNA waspurified with one extraction of equilibrated phenol (pH>8.0, UnitedStates Biochemical, Cleveland, Ohio)-chloroform-isoamyl alcohol(50:49:1) and one extraction with chloroform:isoamyl alcohol (49:1).After the extractions, the DNA was ethanol precipitated and pelleted asdescribed above. The DNA pellets were dried, then dissolved in 50 μLsterile water. The concentration was determined by measuring theabsorbance of an aliquot of the DNA at 260 nm using 33 μg/mL for anabsorbance of 1.0. Samples were stored at −20° C.

Example 5 Antibody Phage Display Vector

The antibody phage display vector for cloning antibodies was derivedfrom an M13 vector supplied by Ixsys, designated 668-4. The vector 668-4contained the DNA sequences encoding the heavy and light chains of amouse monoclonal Fab fragment inserted into a vector described by Huse,WO 92/06024. The vector had a Lac promoter, a pelB signal sequence fusedto the 5′ side of the L chain variable region of the mouse antibody, theentire kappa chain of the mouse antibody, an alkaline phosphatase signalsequence at the 5′ end of the H chain variable region of the mouseantibody, the entire variable region and the first constant region ofthe H chain, and 5 codons of the hinge region of an IgGl H chain. Adecapeptide sequence was at the 3′ end of the H chain hinge region andan amber stop codon separated the decapeptide sequence from thepseudo-gene VIII sequence. The amber stop allowed expression of H chainfusion proteins with the gene VIII protein in E. coli suppressor strainssuch as XL1 blue (STRATAGENE™ Corporation, San Diego, Calif.), but notin nonsuppressor cell strains such as MK3O (Boehringer Mannheim,Indianapolis, Ind.) (see FIG. 3A).

To make the first derivative cloning vector, deletions were made in thevariable regions of the H chain and the L chain by oligonucleotidedirected mutagenesis of a uracil template (Kunkel, Proc. Natl. Acad.Sci. USA 82, 488 (1985); Kunkel et al., Methods. Enzymol. 154, 367(1987)). These mutations deleted the region of each chain from the 5′end of CDR1 to the 3′ end of CDR3, and the mutations added a DNAsequence where protein translation would stop (see FIG. 4 formutagenesis oligonucleotides). This prevented the expression of H or Lchain constant regions in clones without an insert, thereby allowingplaques to be screened for the presence of insert. The resulting cloningvector was called BS11.

Many changes were made to BS11 to generate the cloning vector used inthe present screening methods. The amber stop codon between the heavychain and the pseudo gene VIII sequence was removed so that every heavychain was expressed as a fusion protein with the gene VIII protein. Thisincreased the copy number of the antibodies on the phage relative toBS11. A HindIII restriction enzyme site in the sequence between the 3′end of the L chain and the 5′ end of the alkaline phosphatase signalsequence was deleted so antibodies could be subcloned into a pBR322derivative (Example 13). The interchain cysteine residues at thecarboxy-terminus of the L and H chains were changed to serine residues.This increased the level of expression of the antibodies and the copynumber of the antibodies on the phage without affecting antibodystability. Nonessential DNA sequences on the 5′ side of the lac promoterand on the 3′ side of the pseudo gene VIII sequence were deleted toreduce the size of the M13 vector and the potential for rearrangement. Atranscriptional stop DNA sequence was added to the vector at the L chaincloning site to replace the translational stop so that phage with onlyheavy chain proteins on their surface, which might be nonspecifically inpanning, could not be made. Finally, DNA sequences for protein tags wereadded to different vectors to allow enrichment for polyvalent phage bymetal chelate chromatography (polyhistidine sequence) or by affinitypurification using a decapeptide tag and a magnetic latex having animmobilized antibody that binds the decapeptide tag. The vector BS39 hada polyhistidine sequence at the 3′ end of the kappa chain with no tag atthe end of the heavy chain (FIG. 3B) BS45 had a polyhistidine sequencebetween the end of the heavy chain constant region and the pseudo-geneVIII sequence, and a decapeptide sequence at the 3′ end of the kappachain constant region (FIG. 3C).

Example 6 Preparation of Uracil Templates Used in Generation of SpleenAntibody Phage Libraries

1 mL of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight culture wasadded to 50 ml 2×YT in a 250 mL baffled shake flask. The culture wasgrown at 37° C. to OD₆₀₀=0.6, inoculated with 10 μl of a 1/100 dilutionof vector phage stock and growth continued for 6 hr. Approximately 40 mLof the culture was centrifuged at 12 krpm for 15 minutes at 4° C. Thesupernatant (30 mL) was transferred to a fresh centrifuge tube andincubated at room temperature for 15 minutes after the addition of 15 μlof 10 mg/ml RnaseA (Boehringer Mannheim, Indianapolis, Ind.). The phagewere precipitated by the addition of 7.5 ml of 20% polyethylene glycol8000 (Fisher Scientific, Pittsburgh, Pa.)/3.5M ammonium acetate (SigmaChemical Co., St. Louis, Mo.) and incubation on ice for 30 min. Thesample was centrifuged at 12 krpm for 15 min at 2-8° C. The supernatantwas carefully discarded, and the tube was briefly spun to remove alltraces of supernatant. The pellet was resuspended in 400 μl of high saltbuffer (300 mM NaCl, 100 mM Tris pH 8.0, 1 mM EDTA), and transferred toa 1.5 mL tube. The phage stock was extracted repeatedly with an equalvolume of equilibrated phenol:chloroform:isoamyl alcohol (50:49:1) untilno trace of a white interface was visible, and then extracted with anequal volume of chloroform:isoamyl alcohol (49:1). The DNA wasprecipitated with 2.5 volumes of ethanol and 1/5 volume 7.5 M ammoniumacetate and incubated 30 min at −20° C. The DNA was centrifuged at 14krpm for 10 min at 4° C., the pellet washed once with cold 70% ethanol,and dried in vacuo. The uracil template DNA was dissolved in 30 μlsterile water and the concentration determined by A₂₆₀ using anabsorbance of 1.0 for a concentration of 40 μg/ml. The template wasdiluted to 250 ng/μl with sterile water, aliquoted, and stored at −20°C.

Example 7 Mutagenesis of Uracil Template With ss-DNA and ElectroporationInto E. coli to Generate Antibody Phage Libraries

Antibody phage-display libraries were generated by simultaneouslyintroducing single-stranded heavy and light chain genes onto aphage-display vector uracil template. A typical mutagenesis wasperformed on a 2 μg scale by mixing the following in a 0.2 mL PCRreaction tube: 8 μl of (250 ng/μl) uracil template (examples 5 and 6), 8μl of 10×annealing buffer (200 mM Tris pH 7.0, 20 mM MgCl₂, 500 mMNaCl), 3.33 μl of kinased single-stranded heavy chain insert (100 ng/μl), 3.1 μl of kinased single-stranded light chain insert (100 ng/ml), andsterile water to 80 μl. DNA was annealed in a GeneAmp® 9600 thermalcycler using the following thermal profile: 20 sec at 94° C., 85° C. for60 sec, 85° C. to 55° C. ramp over 30 min, hold at 55° C. for 15 min.The DNA was transferred to ice after the program finished. Theextension/ligation was carried out by adding 8 μl of 10×synthesis buffer(5 mM each dNTP, 10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl₂, 20 mM DTT),8 μl T4 DNA ligase (1U/μl, Boehringer Mannheim, Indianapolis, Ind.), 8μl diluted T7 DNA polymerase (1U/μl, New England BioLabs, Beverly,Mass.) and incubating at 37° C. for 30 min. The reaction was stoppedwith 300 μl of mutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA).The mutagenesis DNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μl of sterile water. 1 μlmutagenesis DNA was (500 ng) was transferred into 40 μl electrocompetentE. coli DH12S (Gibco/BRL, Gaithersburg, Md.) using the electroporationconditions in Example 8. The transformed cells were mixed with 1.0 mL2×YT broth (Sambrooke et al., supra) and transferred to 15 mL sterileculture tubes. The first round antibody phage was made by shaking thecultures overnight at 23° C. and 300 rpm. The efficiency of theelectroporation was measured by plating 10 μl of 10⁻³ and 10⁻⁴ dilutionsof the cultures on LB agar plates (see Example 12). These plates wereincubated overnight at 37° C. The efficiency was determined bymultiplying the number of plaques on the 10⁻³ dilution plate by 10⁵ ormultiplying the number of plaques on the 10⁻⁴ dilution plate by 10⁶. Theovernight cultures from the electroporations were transferred to 1.5 mltubes, and the cells were pelleted by centrifuging at 14 krpm for 5 min.The supernatant, which is the first round of antibody phage, was thentransferred to 15 mL sterile centrifuge tubes with plug seal caps.

Example 8 Transformation of E. coli by Electroporation

The electrocompetent E. coli cells were thawed on ice. DNA was mixedwith 20-40 μL electrocompetant cells by gently pipetting the cells upand down 2-3 times, being careful not to introduce air-bubble. The cellswere transferred to a Gene Pulser cuvette (0.2 cm gap, BioRAD, Hercules,Calif.) that had been cooled on ice, again being careful not tointroduce an air-bubble in the transfer. The cuvette was placed in theE. coli Pulser (BioRAD, Hercules, Calif.) and electroporated with thevoltage set at 1.88 kV according to the manufacturer's recommendation.The transformed sample was immediately diluted to 1 ml with 2×YT brothand processed as procedures dictate.

Example 9 Preparation of Biotinylated Antigens and Antibodies

Protein antigens or antibodies were dialyzed against a minimum of 100volumes of 20 mM borate, 150 mM NaCl, pH 8 (BBS) at 2-8° C. for at least4 hr. The buffer was changed at least once prior to biotinylation.Protein antigens or antibodies were reacted with biotin-XX-NHS ester(Molecular Probes, Eugene, Oreg., stock solution at 40 mM indimethylformamide) at a final concentration of 1 mM for 1 hr at roomtemperature. After 1 hr, the protein antigens or antibodies wereextensively dialyzed into BBS to remove unreacted small molecules.

Example 10 Preparation of Alkaline Phosphatase-antigen Conjugates

Alkaline phosphatase (AP, Calzyme Laboratories, San Luis Obispo, Calif.)was placed into dialysis versus a minimum of 100 volumes of columnbuffer (50 mM potassium phosphate, 10 mM borate, 150 mM NaCl, 1 mMMgSO₄, pH 7.0) at 2-8° C. for at least four hr. The buffer was changedat least twice prior to use of the AP. When the AP was removed fromdialysis and brought to room temperature, the concentration wasdetermined by absorbance at 280 nm using an absorbance of 0.77 for a 1mg/mL solution. The AP was diluted to 5 mg/mL with column buffer. Thereaction of AP and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Pierce Chemical Co., Rockford, Ill.)was carried out using a 20:1 ratio of SMCC:AP. SMCC was dissolved inacetonitrile at 20 mg/mL and diluted by a factor of 84 when added to APwhile vortexing or rapidly stirring. The solution was allowed to standat room temperature for 90 min before the unreacted SMCC and lowmolecular weight reaction products were separated from the AP using gelfiltration chromatography (G50 Fine, Pharmacia Biotech, Piscataway,N.J.) in a column equilibrated with column buffer.

Protein antigen was dialyzed versus a minimum of 100 volumes of 20 mMpotassium phosphate, 4 mM borate, 150 mM NaCl, pH 7.0 at 2-8° C. for atleast four hr. The buffer was changed at least twice prior to use of theantigen. The amount of antigen was quantitated by absorbance at 280 nm.The extinction coefficient for creatine kinase MB subunits (CKMB,Scripps Laboratories, San Diego, Calif.) was 0.88 mL/mg-cm, Clostridiumdifficile toxin A (Tech Lab, Blacksburg, Va.) was 1.29 mL/mg-cm, andClostridium difficile glutamate dehydrogenase (Example 19) was 1.45mL/mg-cm. The reaction of antigen and N-succinimidyl3-[2-pyridyldithio]propionate (SPDP, Pierce Chemical Co., Rockford,Ill.) was carried out using a 20:1 molar ratio of SPDP:antigen. SPDP wasdissolved in dimethylformamide at 40 mM and diluted into the antigensolution while vortexing. The solution was allowed to stand at roomtemperature for 90 min, at which time the reaction was quenched byadding taurine (Aldrich Chemical Co., Milwaukee, Wis.) to a finalconcentration of 20 mM for 5 min. Dithiothreitol (Fisher Scientific,Pittsburgh, Pa.) was added to the protein at a final concentration of 1mM for 30 min. The low molecular weight reaction products were separatedfrom the antigen using gel filtration chromatography in a columnequilibrated in 50 mM potassium phosphate, 10 mM borate, 150 mM NaCl,0.1 mM ethylene diamine tetraacetic acid (EDTA, Fisher Scientific,Pittsburgh, Pa.), pH 7.0.

The AP and antigen were mixed together in an equimolar ratio. Thereaction was allowed to proceed at room temperature for 2 hr. Theconjugate was diluted to 0.1 mg/mL with block containing 1% bovine serumalbumin (from 30% BSA, Bayer, Kankakee, Ill.), 10 mM Tris, 150 mM NaCl,1 mM MgCl₂, 0.1 mM ZnCl₂, 0.1% polyvinyl alcohol (80% hydrolyzed,Aldrich Chemical Co., Milwaukee, Wis.), pH 8.0.

Example 11 Preparation of Avidin Magnetic Latex

The magnetic latex (Estapor, 10% solids, Bangs Laboratories, Fishers,Ind.) was thoroughly resuspended and 2 ml aliquoted into a 15 ml conicaltube. The magnetic latex was suspended in 12 ml distilled water andseparated from the solution for 10 min using a magnet. While still inthe magnet, the liquid was carefully removed with a 10 mL sterile pipet.This washing process was repeated an additional three times. After thefinal wash, the latex was resuspended in 2 ml of distilled water. In aseparate 50 ml conical tube, 10 mg of avidin-HS (NeutrAvidin, Pierce,Rockford, Ill.) was dissolved in 18 ml of 40 mM Tris, 0.15 M sodiumchloride, pH 7.5 (TBS). While vortexing, the 2 ml of washed magneticlatex was added to the diluted avidin-HS and the mixture vortexed anadditional 30 seconds. This mixture was incubated at 45° C. for 2 hr,shaking every 30 minutes. The avidin magnetic latex was separated fromthe solution using a magnet and washed three times with 20 ml BBS asdescribed above. After the final wash, the latex was resuspended in 10ml BBS and stored at 4° C.

Immediately prior to use, the avidin magnetic latex was equilibrated inpanning buffer (40 mM TRIS, 150 mM NaCl, 20 mg/mL BSA, 0.1% Tween 20(Fisher Scientific, Pittsburgh, Pa.), pH 7.5). The avidin magnetic latexneeded for a panning experiment (200 μl/sample) was added to a sterile15 ml centrifuge tube and brought to 10 ml with panning buffer. The tubewas placed on the magnet for 10 min to separate the latex. The solutionwas carefully removed with a 10 mL sterile pipet as described above. Themagnetic latex was resuspended in 10 mL of panning buffer to begin thesecond wash. The magnetic latex was washed a total of 3 times withpanning buffer. After the final wash, the latex was resuspended inpanning buffer to the initial aliquot volume.

Example 12 Plating M13 Phage or Cells Transformed With AntibodyPhage-display Vector Mutagenesis Reaction

The phage samples were added to 200 μL of an overnight culture of E.coli XL1-Blue when plating on 100 mm LB agar plates or to 600 μL ofovernight cells when plating on 150 mm plates in sterile 15 ml culturetubes. After adding LB top agar (3 mL for 100 mm plates or 9 mL for 150mm plates, top agar stored at 55° C., Appendix A1, Molecular Cloning, ALaboratory Manual, (1989) Sambrook. J), the mixture was evenlydistributed on an LB agar plate that had been pre-warmed (37° C.-55° C.)to remove any excess moisture on the agar surface. The plates werecooled at room temperature until the top agar solidified. The plateswere inverted and incubated at 37° C. as indicated.

Example 13 Develop Nitrocellulose Filters With Alkaline PhosphataseConjugates

After overnight incubation of the nitrocellulose filters on LB agarplates, the filters were carefully removed from the plates with membraneforceps and incubated for 2 hr in either casein block (block with 1%casein (Hammersten grade, Research Organics, Cleveland, Ohio)), whenusing antigen-AP conjugates or block when using goat anti-mouse kappa-AP(Southern Biotechnology Associates, Inc, Birmingham, Ala.). After 2 hr,the filters were incubated with the AP conjugate for 2-4 hr. Antigen-APconjugates were diluted into casein block at a final concentration of 1μg/mL and goat anti-mouse kappa-AP conjugates were diluted into block ata final concentration of 1 μg/mL. Filters were washed 3 times with 40 mMTRIS, 150 mM NaCl, 0.05% Tween 20, pH 7.5 (TBST) (Fisher Chemical,Pittsburgh, Pa.) for 5 min each. After the final wash, the filters weredeveloped in a solution containing 0.2 M 2-amino-2-methyl-1-propanol(JBL Scientific, San Luis Obispo, Calif.), 0.5 M TRIS, 0.33 mg/mL nitroblue tetrazolium (Fisher Scientific, Pittsburgh, Pa.) and 0.166 mg/mL5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.

Example 14 Enrichment of Polyclonal Phage to CKMB With No Tags on theHeavy Chain and a Polyhistidine Sequence on the Kappa Chain

This example describes multiple rounds of screening of a phage libraryto the antigen creatine kinase MB (M and B designate muscle and brainsubunits). Some of the rounds of screening to CKMB were alternated withrounds of enrichment for phage displaying multiple copies of antibodies.The percentage of phage displaying any light chain, and the percentageof phage displaying Fab fragments with specific affinity for CKMB wasmeasured after each round of screening to CKMB.

The first round antibody phage was prepared as described above usingBS39 uracil template. Two electroporations of mutagenesis DNA hadefficiencies of 9.7×10⁷ PFU and 8.3×10⁷ PFU. The phage from bothelectroporations were combined and diluted to 3.2 with panning buffer.The phage was aliquoted into 2-1 mL aliquots in 15 mL disposable sterilecentrifuge tubes with plug seal caps. CKMB-biotin (10 μL, 10⁻⁶ M stockconcentration) was added to each phage aliquot. The phage samples wereincubated overnight at 2-8° C. After the incubation, the phage sampleswere panned with avidin magnetic latex. The equilibrated avidin magneticlatex (see Example 11), 200 μL latex per sample, was incubated with thephage for 10 min at room temperature. After 10 min, approximately 9 mLof panning buffer was added to each phage sample, and the magnetic latexwas separated from the solution using a magnet. After 10 min in themagnet, the unbound phage was carefully removed with a 10 mL sterilepipet. The magnetic latex was then resuspended in 10 mL of panningbuffer to begin the second wash. The latex was washed a total of 5 timesas described above. For each wash, the tubes were in the magnet for 10min to separate unbound phage from the magnetic latex. After the 5thwash, the magnetic latex was resuspended in 1 mL TBS and transferred toa 1.5 mL tube. Aliquots of the latex were taken at this point to plateon 100 mm LB agar plates as described above. The bulk of the magneticlatex (99%) was resuspended in 200 μL 2×YT and was plated on a 150 mm LBagar plate as described in Example 12. The 100 mm LB agar plates wereincubated at 37° C. for 6-7 hr, then the plates were transferred to roomtemperature and nitrocellulose filters (pore size 0.45 μm, BA85 Protran,Schleicher and Schuell, Keene, N.H.) were overlayed onto the plaques.Plates with nitrocellulose filters were incubated overnight at roomtemperature. The 150 mm plates were used to amplify the phage binding tothe magnetic latex to generate the next round of antibody phage. Theseplates were incubated at 37° C. for 4 hr, then overnight at 20° C.

After the overnight incubation, the antibody phage was eluted from the150 mm plates, and the filters were developed with alkalinephosphatase-CKMB as described in Example 13. The antibody phage waseluted from the 150 mm plates by pipeting 8 mL 2YT media onto the lawnand gently shaking the plate at room temperature for 20 min. The phagewere transferred to a 15 mL disposable sterile centrifuge tubes withplug seal cap and the debris from the LB plate was pelleted bycentrifuging for 15 min at 3500 rpm. The 2nd round antibody phage wasthen transferred to a new tube.

To begin the 2nd round of panning, the antibody phage were titered byplating 10 μL of 10⁻⁷ and 10⁻⁸ dilutions of the phage on 100 mm LB agarplates. The plates were incubated at 37° C. for 6-7 hr, then the numberof plaques on the plates were counted. Also, to monitor the percentageof kappa positives in the antibody phage, a nitrocellulose filter wasoverlayed onto the plate and incubated overnight at room temperature.The percentage of kappa positives is a measure of the proportion ofphage displaying intact Fab fragments.

Both 2nd round antibody phage samples were pooled by diluting eachsample into panning buffer at a final concentration of 5×10⁹ PFU/mL to afinal volume of 1 mL. (The titers of the antibody phage were about2×10¹² PFU/mL and 1.7×10¹²). CKMB-biotin (10 μL, 10⁻⁶ M stockconcentration) was added to the phage and the phage was incubated at2-8° C. overnight. The nitrocellulose filters on the antibody phagetiter plates were developed with goat anti-mouse kappa AP as describedin Example 13. The second round antibody phage was panned with avidinmagnetic latex as described above. After washing the latex with panningbuffer, the latex was resuspended in 1 mL TBS and transferred to a 1.5mL tube. Aliquots of the latex were plated on 100 mm LB agar plates asdescribed above to check functional positives, and the rest of the latexwas plated on 150 mm LB agar plates to generate the 3rd round antibodyphage. This general procedure of titering the antibody phage, dilutingthe phage into panning buffer and adding CKMB-biotin, incubating thephage at least 16 hr at 2-8° C., panning the phage with avidin magneticlatex, and plating the magnetic latex was followed through 10 rounds ofpanning. The only changes from that described above is the concentrationof CKMB-biotin was lower to increase the affinity of bound antibodies,and the number of phage panned was between 10¹⁰ and 10⁸. The results ofthe filter lifts on the percentage of kappa positives in the antibodyphage and the percentage of functional antibodies binding to themagnetic latex are shown in Table 2.

After the 10th round of panning to CKMB-biotin, the antibody phage weresubject to a round of enrichment for polyvalent display. Enrichment waseffected by binding of the hexahistidine tag fused to the displayedlight chain to NiNTA agarose (Qiagen Inc., Chatsworth, Calif.). The 11thround antibody phage (2.5 mL) were diluted into 2.5 mL panning buffer ina 15 mL disposable sterile centrifuge tube with plug seal cap. The NiNTAwas equilibrated into panning buffer using the following procedure. Theresin (1 mL per phage sample) was diluted to 50 mL with panning bufferin a 50 mL disposable sterile centrifuge tube with plug seal cap andthen was pelleted in an IEC centrifuge at 500 rpm for 1 min. Thesupernatant was carefully removed with a 50 mL disposable pipet, thenthe resin was again diluted to 50 mL with panning buffer for the secondwash. The resin was washed in this manner a total of 4 times in order toequilibrate the resin in panning buffer. The equilibrated resin was thenresuspended to its original volume with panning buffer. Equilibratedresin (1 mL) was then added to the phage, and the tube was gently rockedfor 15 min. After 15 min, the resin was pelleted in an IEC centrifuge at500 rpm for 1 min. The supernatant was gently removed with a 10 mLdisposable pipet, and the resin was resuspended in 10 mL panning bufferfor the first wash. The resin was pelleted as described above, thesupernatant was removed, and the resin was resuspended a 2nd time in 10mL panning buffer. This procedure was repeated for a total of 5 panningbuffer washes. After the 5th wash was removed, the resin was resuspendedin 1 mL of elution buffer (50 mM citrate, 150 mM NaCl, pH 4.0) andtransferred to a 1.5 mL tube. The resin was gently rocked for 1 hr toelute the antibody phage. After 1 hr, the resin was pelleted (14 krpm inEppendorf centrifuge for 5 min), and the phage was removed while beingcareful not to transfer any resin. In order to adjust the pH of thephage solution to 8, 50 μL of 1 M Tris, pH 8.3 and 46 μL of 1 M NaOHwere added to the 1 mL phage sample. Also, 10 μL of 300 mg/mL bovineserum albumin (Bayer, Kankakee, Ill.) was added to the phage sample. Theresulting phage solution (1 mL) was transferred to a 15 mL disposablesterile centrifuge tube with plug seal cap for the 11th round of panningwith CKMB-biotin, as described above. As shown in Table 2, panning theantibody phage with NiNTA prior to panning to CKMB significantlyincreased the percentage of CMB functional positives binding to theavidin magnetic latex.

The 12th-14th rounds of panning were done as described above, where theantibody phage was bound to NiNTA, eluted, and the eluted phage pannedwith CKMB-biotin. However, in round 13, unlabelled creatine kinase BBsubunits (Scripps Laboratories, San Diego, Calif.) and creatine kinaseMM subunits (Scripps Laboratories, San Diego, Calif.) were added to thephage eluted from the NiNTA at 100-fold molar excess to the CKMB-biotinto select antibodies that specifically bind to CKMB without binding toCKMM or CKBB. The percentage of functional CKMB antibodies was greaterthan 95% by round 13.

TABLE 2 Summary of kappa positives and CKMB functional positives at eachround of panning phage binding to antibody phage avidin latex % kapparound # % functional 202/253 (80%) 1 0/32 (0%) 175/234 (75%) 2 0/129(0%) 87/120 (73%) 3 4/79 (5.1%) 81/165 (49%) 4 35/409 (8.6%) 149/342(44%) 5 5/31 (16%) 40/192 (21%) 6 30/400 (7.5%) 1/54 (2%) 7 6/54 (11%)6/106 (6%) 8 4/46 (9%) 35/93 (38%) 9 19/551 (3%) 14/191 (7%) 10 18/400(5%) N/A 11 349/553 (63%) 96/100 (96%) 12 232/290 (80%) 31/31 (100%) 13(>95%) 147/149 (99%) 14 400/405 (99%)

Table 2 illustrates the importance of polyvalent enrichment for makingphage libraries with a high proportion of phage displaying antibodieswith specific affinity for the chosen target (functional positives). Thepercentage of phage with specific affinity for the selected target iszero after the first two rounds of panning but then increase to aboutten percent before levelling off in subsequent rounds of panning throughround ten. Then in rounds 11-14, which are performed concurrently withpolyvalent enrichment, the percentage of functional positive phageincreases 99%. The rapid increase in functional positive phage in rounds11-14, in which polyvalent enrichment is performed, compared with theplateau of about 10% achieved in rounds 1-10 without polyvalentenrichment illustrates the power of the present methods to achievelibraries with a high percent of functional positives.

The variation in percent kappa positives in different rounds ofscreening is also instructive. The percentage is initially high butdecreases in the first ten rounds of screening (which are not performedconcurrently with polyvalent enrichment). The decrease arises becausekappa negative phage grow faster than kappa positive phage and arepreferentially amplified between rounds of panning. In rounds 11-14, thepercent of kappa positives increases to near 100%. The increase is dueto polyvalent selection, which is effected by screening for phagedisplaying at least two kappa chains.

Example 15 Enrichment of Polyclonal Phage to Clostridium difficile ToxinA With a Polyhistidine Tag on the Heavy Chain; Selection of an EpitopeSpecificity on Toxin A Different From a Reference Antibody

The first round antibody phage was prepared as described in Example 7using BS45 uracil template. Eight electroporations of mutagenesis DNAwere performed yielding 8 different phage samples. To create morediversity in the polyclonal library, each phage sample was pannedseparately. The antibody phage (about 0.9 mL) from each electroporationwas transferred to a 15 mL disposable sterile centrifuge tube with plugseal cap. BSA (30 μL 300 mg/mL solution) and 1 M Tris (50 μL, 1 M stocksolution, pH 8.0) were added to each phage stock, followed by 10 μL 10⁻⁶M toxin A-biotin. The antibody phage was allowed to come to equilibriumwith the toxin A-biotin by incubating the phage at 2-8° C. overnight.After the incubation, the phage were panned with avidin magnetic latexas described above in Example 14, except only 4 panning buffer washeswere performed instead of 5. The entire magnetic latex of each samplewas plated on 150 mm LB plates to generate the 2nd round antibody phage.The 150 mm plates were incubated at 37° C. for 4 hr, then overnight at20° C.

The 2nd round antibody phage were eluted from the 150 mm plates asdescribed in Example 14 except 10 mL 2×YT was used to elute the phageinstead of 8 mL. The second round of panning was set up by diluting 100μL of each phage stock (8 total) into 900 μL panning buffer in 15 mLdisposable sterile centrifuge tubes with plug seal cap. Toxin A-biotin(10 μL of 10⁻⁷ M) was added to each sample, and the phage were incubatedovernight at 2-8° C. The antibody phage were not titered before panningas described in Example 14. The phage samples were panned with avidinmagnetic latex following the overnight incubation. After washing thelatexes with panning buffer, each latex was plated on 150 mm LB agarplates. The plates were incubated at 37° C. for 4 hr, then 20° C.overnight.

The 3rd round antibody phage were bound to NiNTA resin and eluted priorto panning with toxin A-biotin. Each phage sample was bound to NiNTA andwashed with panning buffer as described in Example 14. After the finalwash, the antibody phage was eluted by adding 0.8 mL 300 mM imidazole(Fisher Scientific, Pittsburgh, Pa.) in panning buffer to each sample,and rocking the tubes for 10 min at room temperature. The resin waspelleted by centrifuging the tubes at 14 krpm for 5 min at roomtemperature, and the phage were carefully transferred to new tubes. Eachphage sample was diluted to about 1.1 mL with panning buffer, then 1 mLof each sample was transferred to 15 mL disposable sterile centrifugetube with plug seal cap. Toxin A-biotin (10 μL of 10⁻⁷M) was added toeach sample, and the phage were incubated overnight at 2-8° C. After theovernight incubation, the phage were panned with avidin magnetic latex.After washing, each latex was resuspended in 1 mL panning buffer, andaliquots of each latex were plated on 100 mm LB agar plates to determinethe percentage of kappa positives or functional positives. The majorityof latex from each panning (99%) was plated on 150 mm LB agar plates toamplify the phage binding to the latex (see Example 14).

Two nitrocellulose filters from the 3rd round of panning were developedwith goat anti-mouse kappa AP and had 83% and 85% positives. Six filtersdeveloped with toxin A-AP were all between 39-47% functional positive.At this point, 10 μL of 10⁻⁷ and 20 μL of 10⁻⁸ dilutions of eachantibody phage stock were plated on 100 mm LB plates to determine thetiters of each phage stock. The titers of the phage stocks weredetermined prior to pooling so that phage stocks with very high titersdid not bias the antibody phage library. The titers (PFU/mL) weredetermined by counting the number of plaques on each plate andmultiplying that number by 10⁹ (10⁻⁷ dilution plate) or 5×10⁹ (10⁻⁸dilution plate). Once the titers were determined, 2×10¹¹ PFU from eachphage stock was pooled. The pooled phage (1.5 mL) was diluted to 5 mLwith panning buffer, then the phage was panned with NiNTA. The elutedphage was panned to toxin A in the presence of an antibody to toxin A,PCG4 (described U.S. Pat. No. 4,533,630), to screen for phage-antibodiesto toxin A that have a different specificity than PCG4. The eluted phagewas diluted to about 1.1 mL with panning buffer, and 1 mL was aliquotedinto a 15 mL tube. In a 1.5 mL tube, unlabeled toxin A (7.5 μL, 10⁻⁷M)and biotinylated PCG4 (15 μL, 10⁻⁶M) were mixed and incubated at roomtemperature for 15 min. A 20-fold molar excess of biotinylated antibodyover toxin A was used so that no antibody phage could bind at the PCG4epitope. The mixture of toxin A and biotinylated antibody (15 μL) wasadded to the 1 mL phage and incubated overnight at 2-8° C. The phagesample was panned with avidin magnetic latex as described in Example 14.The process of panning the eluted antibody phage, binding and elutingthe phage with NiNTA, and panning the resulting eluted phage withbiotinylated PCG4/toxin A was repeated 4 times. After the 5th round ofselection, the polyclonal antibody phage stock complementary to PCG4 was99% kappa positive and 98% had specific affinity for toxin A by plaquelift analysis.

Example 16 Enrichment of Polyclonal Phage to Clostridium difficileGlutamate Dehydrogenase Using a Decapeptide Tag on the Kappa Chain

The first round antibody phage was prepared as described in Example 7using BS45 uracil template, which has a hexahistidine tag for polyvalentenrichment fused to the heavy chain of displayed Fabs. Twelveelectroporations of mutagenesis DNA from 4 different spleens (3electroporations from each spleen) yielded 12 different phage samples.Each phage sample was panned separately to create more diversity in thepolyclonal library. The antibody phage (about 0.9 mL) from eachelectroporation was transferred to a 15 mL disposable sterile centrifugetube with plug seal cap. BSA (30 μL of a 300 mg/mL solution) and 1 MTris (50 μL, pH 8.0) were added to each phage stock, followed by 10 μL10⁻⁷ M glutamate dehydrogenase-biotin (Example 19). The antibody phagewas allowed to come to equilibrium with the glutamatedehydrogenase-biotin by incubating the phage at room temperature for 4hr. After the incubation, the phage samples were panned with avidinmagnetic latex as described in Example 14. The entire latex of eachsample was plated on 150 mm LB agar plates to generate the 2nd roundantibody phage (Example 14). The resulting phage were panned withglutamate dehydrogenase-avidin-magnetic latex as described for the firstround of panning.

A procedure was developed to enrich the antibody phage using thedecapeptide tag on the kappa chain and a monoclonal antibody magneticlatex that binds the decapeptide. Binding studies had previously shownthat the decapeptide could be eluted from the monoclonal antibody 7F11(see Example 21) at a relatively mild pH of 10.5-11. The third roundantibody phage resulting from panning NiNTA enriched 2nd round phagewere bound to the 7F11 magnetic latex and eluted as described below. The7F11 magnetic latex (2.5 mL) was equilibrated with panning buffer asdescribed above for the avidin magnetic latex. Each phage stock (1 mL)was aliquoted into a 15 mL tube. The 7F11 magnetic latex (200 μL perphage sample) was incubated with phage for 10 min at room temperature.After 10 min, 9 mL of panning buffer was added, and the magnetic latexwas separated from unbound phage by placing the tubes in a magnet for 10min. The latexes were washed with 1 additional 10 mL panning bufferwash. Each latex was resuspended in 1 mL panning buffer and transferredto 1.5 mL tubes. The magnetic latex was separated from unbound phage byplacing the tubes in a smaller magnet for 5 min, then the supernatantwas carefully removed with a sterile pipet. Each latex was resuspendedin 1 mL elution buffer (20 mM 3-(cyclohexylamino)propanesulfonic acid(United States Biochemical, Cleveland, Ohio), 150 mM NaCl, 20 mg/mL BSA,pH 10.5) and incubated at room temperature for 10 min. After 10 min,tubes were placed in the small magnet again for 5 min and the elutedphage was transferred to a new 1.5 mL tube. The phage samples were againplaced in the magnet for 5 min to remove the last bit of latex that wastransferred. Eluted phage was carefully removed into a new tube and 25μL 3 M Tris, pH 6.8 was added to neutralize the phage. Panning withglutamate dehydrogenase-biotin was set up for each sample by mixing 900μL 7F11/decapeptide enriched phage, 100 μL panning buffer, and 10 μL10⁻⁷ M glutamate dehydrogenase-biotin and incubating overnight at 2-8°C. The phage was panned with avidin magnetic latex as described inExample 14.

One of the functional positive plaques was arbitrarily picked off an LBagar plate, and the antibody was subcloned into the expression vectordescribed in Example 18. The monoclonal antibody was used as a referenceto isolate polyclonal antibodies having different epitope specificitythan the monoclonal antibody for sandwich assay development. This issimilar to what is described above for toxin A except the monoclonalantibody for toxin A was a hybridoma antibody from a commercial sourceand the monoclonal antibody described here is from the antibody phagelibrary.

The resulting 4th round antibody phage was panned again with 7F11magnetic latex prior to functional panning. The eluted phage sampleswere set up for panning with the biotinylated monoclonal antibody(CD.43.9) and unlabelled glutamate dehydrogenase as described in Example15 for toxin A antibodies. As discussed above, the monoclonal antibodywas picked from the phage library and a polyclonal antibody phage stockthat was complementary to the monoclonal was needed. Mixed themonoclonal antibody biotin (125 μL, 10⁻⁶M) and glutamate dehydrogenase(125 μL, 5×10⁻⁸ M) at room temperature for 15 min. Added 20 μL of themixture to each 1 mL phage sample (0.7 mL panning buffer and 0.3 mLphage eluted from the 7F11 latex), and incubated the samples overnightat 2-8° C. The phage samples were panned with the avidin magnetic latexfollowing the standard procedure.

The 5th round antibody phage were eluted from the 150 mm LB agar platesas described in Example 15. The antibody phage were titered by plating10 μL 10⁻⁷ dilutions of each phage stock on 100 mm LB agar plates. After6 hr at 37° C., the number of plaques on each plate was counted, and thetiters were calculated by multiplying the number of plaques by 10⁹. Apool of ten fifth round phage was made by mixing an equal number ofphage from each phage stock so that high titer phage stocks would notbias the pool. Two of the samples were discarded because they had lowfunctional percentages by plaque lift. The pooled phage were panned with7F11 magnetic latex as described above. The eluted phage was set up forpanning by mixing 0.1 mL panning buffer, 0.9 mL 7F11 eluted phage, and20 μL monoclonal antibody biotin/glutamate dehydrogenase (see above).Phage were incubated overnight at 2-8° C. The phage sample was pannedwith avidin magnetic latex following the standard procedure. The phagebinding to the latex was 97% functional positive by plaque lift assay.

Example 17 Construction of the pBR Expression Vector

An expression vector and a process for the subcloning of monoclonal andpolyclonal antibody genes from a phage-display vector has been developedthat is efficient, does not substantially bias the polyclonalpopulation, and can select for vector containing an insert capable ofrestoring antibiotic resistance. The vector is a modified pBR322plasmid, designated pBRncoH3, that contains an arabinose promoter,ampicillin resistance (beta-lactamase) gene, a partial tetracyclineresistance gene, a pelB (pectate lyase) signal sequence, and NcoI andHindIII restriction sites. (FIG. 7). The pBRncoH3 vector can also beused to clone proteins other than Fabs with a signal sequence. A secondvector, pBRnsiH3, has been developed for cloning proteins with orwithout signal sequences, identical to the vector described above exceptthat the pelB signal sequence is deleted and the NcoI restriction sitehas been replaced with an NsiI site.

The araC regulatory gene (including the araBAD promoter) was amplifiedfrom E. coli K-12 strain NL31-001 (a gift from Dr. Nancy Lee at UCSB) byPCR (Example 3) using Taq DNA polymerase (Boehringer Mannheim,Indianapolis, Ind.) with primers A and B (Table 3). Primers A and Bcontain 20 base-pairs of the BS39 vector sequence at their 5′-endscomplementary to the 5′ side of the lac promoter and the 5′ side of thepelB signal sequence, respectively. Primer A includes an EcoRIrestriction site at its 5′-end used later for ligating the ara insertinto the pBR vector. The araCparaBAD PCR product was verified by agarosegel electrophoresis and used as template for an asymmetric PCR reactionwith primer ‘B’ in order to generate the anti-sense strand of theinsert. The single-stranded product was run on agarose gelelectrophoresis, excised, purified with GeneClean (Bio101, San Diego,Calif.), and resuspended in water as per manufacturers recommendations.The insert was kinased with T4 polynucleotide kinase for 45 min at 37°C. The T4 polynucleotide kinase was heat inactivated at 70° C. for 10min and the insert extracted with an equal volume of phenol/chloroform,followed by chloroform. The DNA was precipitated with ethanol at −20° C.for 30 min. The DNA was pelleted by centrifugation at 14 krpm for 15 minat 4° C., washed with ice-cold 70% ethanol, and dried in vacuo.

The insert was resuspended in water and the concentration determined byA₂₆₀ using an absorbance of 1.0 for a concentration of 40 μg/ml. Theinsert was cloned into the phage-display vector BS39 for sequenceverification and to introduce the pelB signal sequence in frame with thearabinose promoter (the pelB signal sequence also contains a NcoIrestriction site at its 3′-end used later for ligating the ara insertinto the pBR vector). The cloning was accomplished by mixing 250 ng ofBS39 uracil template (Example 5), 150 ng of kinased araCpBAD insert, and1.0 μl of 10×annealing buffer in a final volume of 10 μl. The sample washeated to 70° C. for 2 min and cooled over 20 min to room temperature toallow the insert and vector to anneal. The insert and vector wereligated together by adding 1 μl of 10×synthesis buffer, 1 μl T4 DNAligase (1U/μl), 1 μl T7 DNA polymerase (1 U/μl) and incubating at 37° C.for 30 min. The reaction was stopped with 90 μl of stop buffer (10 mMTris pH 8.0, 10 mM EDTA) and 1 μl electroporated (Example 8) intoelectrocompetent E. coli strain, DH10B, (Life Technologies,Gaithersburg, Md.).

The transformed cells were diluted to 1.0 ml with 2×YT broth and 1 μl,10 μl, 100 μl plated as described in Example 12. Following incubationovernight at 37° C., individual plaques were picked, amplified by PCRwith primers A and B, and checked for full-length insert by agarose gelelectrophoresis. Clones with full-length insert were sequenced withprimers D, E , F, G (Table 3) and checked against the literature. Aninsert with the correct DNA sequence was amplified by PCR (Example 3)from BS39 with primers A and C (FIG. 5A) and the products run on agarosegel electrophoresis.

Full-length products were excised from the gel and purified as describedpreviously and prepared for cloning by digestion with EcoRI and NcoI. ApBR lac-based expression vector that expressed a murine Fab was preparedto receive this insert by EcoRI and NcoI digestion. This digestionexcised the lac promoter and the entire coding sequence up to the 5′-endof the heavy chain (CH1) constant region (FIG. 5A).

The insert and vector were mixed (2:1 molar ratio) together with 1 μl 10mM ATP, 1 μl (1U/μl) T4 DNA ligase, 1 μl 10×ligase buffer in a finalvolume of 10 μl and ligated overnight at 15° C. The ligation reactionwas diluted to 20 μl, and 1 μl electroporated into electrocompetent E.coli strain, DH10B (Example 8), plated on LB tetracycline (10 μg/ml)plates and grown overnight at 37° C.

Clones were picked and grown overnight in 3 ml LB broth supplementedwith tetracycline at 20 μg/ml. These clones were tested for the correctinsert by PCR amplification (Example 3) with primers A and C, using 1 μlof overnight culture as template. Agarose gel electrophoresis of the PCRreactions demonstrated that all clones had the araCparaB insert. Thevector (plasmid) was purified from each culture by Wizard miniprepcolumns (Promega, Madison, Wis.) following manufacturersrecommendations. The new vector contained the araC gene, the araBpromoter, the pelB signal sequence, and essentially the entire CH1region of the heavy chain (FIG. 5B).

The vector was tested for expression by re-introducing the region of theFab that was removed by EcoRI and NcoI digestion. The region wasamplified by PCR, (Example 3) from a plasmid (20 ng) expressing 14F8with primers H and I (Table 3). The primers, in addition to havingsequence specific to 14F8, contain 20 base-pairs of vector sequence attheir 5′-end corresponding to the 3′-end of the pelB signal sequence andthe 5′-end of the CH1 region for cloning purposes. The PCR products wererun on agarose gel electrophoresis and full-length products excised fromthe gel and purified as described previously.

The vector was linearized with NcoI and together with the insert,prepared for cloning through the 3′→5′ exonuclease activity of T4 DNApolymerase. The insert and NcoI digested vector were prepared for T4exonuclease digestion by aliquoting 1.0 μg of each in separate tubes,adding 1.0 μl of 10×restriction endonuclease Buffer A (BoehringerMannheim, Indianapolis, Ind.) and bringing the volume to 9.0 μl withwater. The samples were digested for 5 min at 30° C. with 1 μl (1U/μl)of T4 DNA polymerase. The T4 DNA polymerase was heat inactivated byincubation at 70° C. for 15 min. The samples were cooled, briefly spun,and the digested insert (35 ng) and vector (100 ng) mixed together andthe volume brought to 10 μl with 1 mM MgCl₂. The sample was heated to70° C. for 2 min and cooled over 20 min to room temperature to allow thecomplementary 5′ single-stranded overhangs of the insert and vectorresulting from the exonuclease digestion to anneal together (FIG. 6).The annealed DNA (1.5 μl) was electroporated (Example 8) into 30 μl ofelectrocompetent E. coli strain DH10B.

The transformed cells were diluted to 1.0 ml with 2×YT broth and 1 μl,10 μl, and 100 μl plated on LB agar plates supplemented withtetracycline (10 μg/ml) and grown overnight at 37° C. The following day,two clones were picked and grown overnight in 2×YT (10 μg/mltetracycline) at 37° C. To test protein expression driven from the arapromoter, these cultures were diluted 1/50 in 2×YT(tet) and grown toOD₆₀₀=1.0 at which point they were each split into two cultures, one ofwhich was induced by the addition of arabinose to a final concentrationof 0.2% (W/V). The cultures were grown overnight at room temperature,and assayed for Fab production by ELISA. Both of the induced cultureswere producing approximately 20 μg/ml Fab. There was no detectable Fabin the uninduced cultures.

Initial efforts to clone polyclonal populations of Fab were hindered bybackgrounds of undigested vector ranging from 3-13%. This undigestedvector resulted in loss of Fab expressing clones due to the selectiveadvantage non-expressing clones have over Fab expressing clones. Avariety of means were tried to eliminate undigested vector from thevector preparations with only partial success; examples including:digesting the vector overnight 37° C. with NcoI, extracting, andredigesting the preparation a second time; including spermidine in theNcoI digest; including single-stranded binding protein (United StatesBiochemical, Cleveland, Ohio) in the NcoI digest; preparative gelelectrophoresis. It was then noted that there is a HindIII restrictionsite in pBR, 19 base-pairs from the 5′-end of the tetracycline promoter.A vector missing these 19 base-pairs is incapable of supporting growthin the presence of tetracycline, eliminating background due toundigested vector.

The ara-based expression vector was modified to make it tetracyclinesensitive in the absence of insert. This was done by digesting thepBRnco vector with NcoI and HindIII (Boehringer Mannheim, Indianapolis,Ind.), which removed the entire antibody gene cassette and a portion ofthe tet promoter (FIG. 5B). The region excised by NcoI/HindIII digestionwas replaced with a stuffer fragment of unrelated DNA by ligation asdescribed above. The ligation reaction was diluted to 20 μl, and 1 μlelectroporated (Example 8) into electrocompetent E. coli strain DH10B,plated on LB ampicillin (100 μg/ml) and incubated at 37° C.

After overnight incubation, transformants were picked and grownovernight in LB broth supplemented with ampicillin (100 μg/ml). Thevector (plasmid) was purified from each culture by Wizard miniprepcolumns following manufacturers recommendations. This modified vector,pBRncoH3, is tet sensitive, but still retains ampicillin resistance forgrowing preparations of the vector.

The antibody gene inserts were amplified by PCR with primers I and J(Table 3) as described in Example 3; primer J containing the 19base-pairs of the tet promoter removed by HindIII digestion, in additionto 20 base-pairs of vector sequence 3′ to the HindIII site forannealing. This modified vector was digested with NcoI/HindIII and,together with the insert, exonuclease digested and annealed as describedpreviously. The tet resistance is restored only in clones that containan insert capable of completing the tet promoter. The annealedFab/vector (1 μl) was transformed (Example 8) into 30 μl ofelectrocompetent E. coli strain, DH10B.

The transformed cells were diluted to 1.0 ml with 2×YT broth and 10 μlof 10⁻² and 10⁻³ dilutions plated on LB agar plates supplemented withtetracycline at 10 μg/ml to determine the size of the subclonedpolyclonal population. This plating also provides and opportunity topick individual clones from the polyclonal if necessary. The remainingcells were incubated at 37° C. for 1 hr and then diluted 1/100 into 30ml 2×YT supplemented with 1% glycerol and 20 μg/ml tetracycline andgrown overnight at 37° C. The overnight culture was diluted 1/100 intothe same media and grown 8 hr at which time glycerol freezer stocks weremade for long term storage at −80° C.

The new vector eliminates growth bias of clones containing vector only,as compared to clones with insert. This, together with the arabinosepromoter which is completely repressed in the absence of arabinose,allows cultures of transformed organisms to be expanded without biasingthe polyclonal antibody population for antibodies that are bettertolerated by E. coli until induction.

A variant of this vector was also constructed to clone any protein withor without a signal sequence. The modified vector has the NcoIrestriction site and all of the pelB signal-sequence removed. In itsplace a NsiI restriction site was incorporated such that upon NsiIdigestion and then T4 digestion, there is single base added, in frame,to the araBAD promoter that becomes the adenosine residue (A) of the ATGinitiation codon. The HindIII site and restoration of the tetracyclinepromoter with primer J (Table 3) remains the same as described for thepBRncoH3 vector. Additionally, the T4 exonuclease cloning process isidentical to that described above, except that the 5′ PCR primer used toamplify the insert contains 20 bp of vector sequence at its 5′-endcorresponding to 3′-end of the araBAD promoter rather than the 3′-end ofthe PelB signal sequence.

Three PCR primers, K, L, and M (Table 3) were used for amplifying thearaC regulatory gene (including the araBAD promoter). The 5′-primer,primer K, includes an EcoRI restriction site at its 5′-end for ligatingthe ara insert into the pBR vector. The 3′-end of the insert wasamplified using two primers because a single primer would have been toolarge to synthesize. The inner 3′-primer (L) introduces the NsiIrestriction site, in frame, with the araBAD promoter, with the outer 3′primer (M) introducing the HinDIII restriction site that will be usedfor ligating the insert into the vector.

The PCR reaction was performed as in Example 3 on a 4×100 μl scale; thereactions containing 100 pmol of 5′ primer (K), 1 pmol of the inner 3′primer (L), and 100 pmol of outer 3′ primer (M), 10 μl 2 mM dNTPs, 0.5μL Taq DNA Polymerase, 10 μl 10×Taq DNA polymerase buffer with MgCl₂,and H₂O to 100 μL. The araCparaBAD PCR product was precipitated andfractionated by agarose gel electrophoresis and full-length productsexcised from the gel, purified, resuspended in water, and prepared forcloning by digestion with EcoRI and HinDIII as described earlier. ThepBR vector (Life Technologies, Gaithersburg, Md.) was prepared toreceive this insert by digestion with EcoRI and HindIII and purificationby agarose gel electrophoresis as described above.

The insert and vector were mixed (2:1 molar ratio) together with 1 μl 10mM ATP, 1 μl (1 U/μl) T4 DNA ligase, 1 μl 10×ligase buffer in a finalvolume of 10 μl and ligated overnight at 15° C. The ligation reactionwas diluted to 20 μl, and 1 μl electroporated into electrocompetent E.coli strain, DH10B (Example 8), plated on LB tetracycline (10 μg/ml)plates and grown overnight at 37° C. Clones were picked and grownovernight in 3 ml LB broth supplemented with tetracycline.

These clones were tested for the correct insert by PCR amplification(Example 3) with primers K and M, using 1 μl of overnight culture astemplate. Agarose gel electrophoresis of the PCR reactions demonstratedthat all clones had the araCparaB insert. The vector (plasmid) waspurified from each culture by Wizard miniprep columns followingmanufacturers recommendations. The new vector, pBRnsi contained the araCgene, the araBAD promoter, and a NsiI restriction site.

The vector was tested for expression by introducing a murine Fab. Theregion was amplified by PCR (Example 3) from a plasmid (20 ng)containing a murine Fab with primers O and N (Table 3). The primers, inaddition to having sequence specific to the Fab, contain 20 bp of vectorsequence at their 5′-end corresponding to the 3′-end araBAD promoter andthe 5′-end of the CH1 region for cloning purposes The pBRnsi vector waslinearized with NsiI and HindIII. The vector and the PCR product wererun on an agarose gel, and full-length products were excised from thegel and purified as described previously. The vector and insert weredigested with T4 DNA polymerase and annealed as described earlier. Theannealed DNA (1 μl) was electroporated (Example 8) into 30 μl ofelectrocompetent E. coli strain DH10B. The transformed cells werediluted to 1.0 ml with 2×YT broth and 1 μl, 10 μl, and 100 μl plated onLB agar plates supplemented with tetracycline (10 μg/ml) and grownovernight at 37° C.

Nitrocellulose lifts were placed on the placed on the surface of theagar plates for 1 min and processed as described (Section 12.24,Molecular Cloning, A laboratory Manual, (1989) Sambrook. J.). Thefilters were developed with goat anti-kappa-AP, and a positive (kappaexpressing) clone was picked and grown overnight in 2×YT (10 μg/mltetracycline) at 37° C. The vector (plasmid) was purified from theculture by Wizard miniprep columns (Promega, Madison, Wis.) followingmanufacturers recommendations. The Fab region was excised byNcoI/HindIII digestion and replaced with a stuffer fragment of unrelatedDNA by ligation as described above. The ligation reaction was diluted to20 μl, and 1 μl electroporated (Example 8) into electrocompetent E. colistrain DH10B, plated on LB ampicillin (100 μg/ml) and incubated at 37°C. After overnight incubation, transformants were picked and grownovernight in LB broth supplemented with ampicillin (100 μg/ml). Thevector (plasmid) was purified from each culture by Wizard miniprepcolumns following manufacturers recommendations. This modified vector,pBRnsiH3, is tet sensitive, but still retains ampicillin resistance forgrowing preparations of the vector.

Example 18 Subcloning Monoclonal and Polyclonal Fab Populations IntoExpression Vectors and Electroporation Into Escherichia coli

The final round of the polyclonal glutamate dehydrogenase antibody phage(see Example 16) was diluted 1/30 in 2×YT (approximately 2×10⁹/ml) and 1μl used as template for PCR amplification of the antibody gene insertswith primers I and P (Table 3). PCR (3-100 μL reactions) was performedusing a high-fidelity PCR system, Expand (Boehringer Mannheim,Indianapolis, Ind.) to minimize errors incorporated into the DNAproduct. Each 100 μl reaction contained 100 pmol of 5′ primer I, 100pmol of 3′ primer J, 0.7 units of Expand DNA polymerase, 10 μl 2 mMdNTPs, 10 μl 10×Expand reaction buffer, 1 μl diluted phage stock astemplate, and water to 100 μl. The reaction was carried out in aPerkin-Elmer thermal cycler (Model 9600) using the following thermalprofile: one cycle of denaturation at 94° C. (1 min); ten cycles ofdenaturation (15 sec, 94° C.), annealing (30 sec, 55° C.), elongation(60 sec, 72° C.); fifteen cycles of denaturation (15 sec, 94° C.),annealing (30 sec, 55° C.), elongation (80 sec plus 20 sec for eachadditional cycle, 72° C.); elongation (6 min, 72° C.); soak (4° C.,indefinitely). The PCR products were ethanol precipitated, pelleted anddried as described above. The DNA was dissolved in water andfractionated by agarose gel electrophoresis. Only full-length productswere excised from the gel, purified, and resuspended in water asdescribed earlier.

The insert and NcoI/HindIII digested pBRncoH3 vector were prepared forT4 exonuclease digestion by adding 1.0 μl of 10×Buffer A to 1.0 μg ofDNA and bringing the final volume to 9 μl with water. The samples weredigested for 4 min at 30° C. with 1 μl (1U/μl) of T4 DNA polymerase. TheT4 DNA polymerase was heat inactivated by incubation at 70° C. for 10min. The samples were cooled, briefly spun, and 5 μl of the digestedantibody gene insert and 2.0 μl of 10×annealing buffer were mixed with 5μL of digested vector in a 1.5 mL tube. The volume was brought to 20 μlwith water, heated to 70° C. for 2 min and cooled over 20 min to roomtemperature to allow the insert and vector to anneal.

The insert and vector were ligated together by adding 2 μl of10×synthesis buffer, 2 μl T4 DNA ligase (1U/μl), 2 μl diluted T7 DNApolymerase (1U/μl) and incubating at 37° C. for 30 min. The reaction wasstopped with 370 μl of stop buffer (10 mM Tris pH 8.0, 10 mM EDTA),extracted with phenol/chloroform, chloroform, and precipitated fromethanol at −20° C. The reaction was centrifuged and the supernatantaspirated. The sample was briefly spun an additional time and all tracesof ethanol removed with a pipetman. The pellet was dried in vacuo.

The DNA was resuspended in 2 μl of water and 1 μl electroporated(Example 8) into 40 μl of electrocompetent E. coli strain, DH10B. Thetransformed cells were diluted to 1.0 ml with 2×YT broth and 10 μl of10⁻¹, 10⁻² and 10⁻³ dilutions plated on LB agar plates supplemented withtetracycline at 10 μg/ml to determine the size of the subclonedpolyclonal population. The remaining cells were incubated at 37° C. for1 hr and then diluted 1/100 into 30 ml 2×YT supplemented with 1%glycerol and 20 μg/ml tetracycline and grown overnight at 37° C. Theovernight culture was diluted 1/100 into the same media, grown 8 hr, andglycerol freezer stocks made for long term storage at −80° C. Thepolyclonal antibody was designated CD.43.5.PC.

The monoclonal antibody to glutamate dehydrogenase (Example 16) was alsosubcloned following the same general procedure described above. Thesubcloned monoclonal antibody was designated CD.43.9. The polyclonalantibody phage stock for Clostridium difficile toxin A (Example 15) wassubcloned in a similar way. The subcloned polyclonal antibody wasdesignated CD.TXA.1.PC.

Example 19 Cloning of Clostridium difficile Glutamate Dehydrogenase (theTarget for Panning in Example 15)

PCR primers were made corresponding to the coding sequence at the 5′-endof glutamate dehydrogenase, and the coding sequence at the 3′-end ofglutamate dehydrogenase, including six histidine codons inserted betweenthe end of the coding sequence and the stop codon to assist inpurification of the recombinant protein by metal-chelate chromatography,primers Q and R, respectively (Table 3). In addition, the 5′ primercontains 20 base-pairs of vector sequence at its 5′-end corresponding tothe 3′-end of the pBRnsiH3 vector. The 3′ primer contains the 19base-pairs of tet promoter removed by HindIII digestion, in addition to20 base-pairs of vector sequence 3′ to the HindIII site at its 5′ end(Example 17).

The PCR amplification of the glutamate dehydrogenase gene insert wasdone on a 100 μl reaction scale containing 100 pmol of 5′ primer (Q),100 pmol of 3′ primer (R), 2 units of Expand polymerase, 10 μl 2 mMdNTPs, 10 μl 10×Expand reaction buffer, 1 μl C. difficile genomic DNA(75 ng) as template, and water to 100 μl. The reaction was carried outin a Perkin-Elmer thermal cycler as described in Example 18. The PCRproducts were precipitated and fractionated by agarose gelelectrophoresis and full-length products excised from the gel, purified,and resuspended in water (Example 17). The insert and NsiI/HindIIIdigested pBRnsiH3 vector were prepared for T4 exonuclease digestion byadding 1.0 μl of 10×Buffer A to 1.0 μg of DNA and bringing the finalvolume to 9 μl with water. The samples were digested for 4 min at 30° C.with 1 μl (1U/μl) of T4 DNA polymerase. The T4 DNA polymerase was heatinactivated by incubation at 70° C. for 10 min. The samples were cooled,briefly spun, and 70 ng of the digested insert added to 100 ng ofdigested pBRnsiH3 vector in a fresh microfuge tube. After the additionof 1 μl of 10×annealing buffer, the volume was brought to 10 μl withwater and the mixture heated to 70° C. for 2 min and cooled over 20 minto room temperature. The annealed DNA was diluted one to four withdistilled water and electroporated (Example 8) into 30 μl ofelectrocompetent E. coli strain, DH10B. The transformed cells werediluted to 1.0 ml with 2×YT broth and 10 μl, 100 μl and 300 μl plated onLB agar plates supplemented with tetracycline (10 μg/ml) and grownovernight at 37° C. Clones were picked and grown overnight in 2×YT (10μg/ml tetracycline) at 37° C. The following day glycerol freezer stocksmade for long term storage at −80° C. The glutamate dehydrogenase clonewas grown and purified on a preparative scale as described in Example20.

Example 20 Growth of E. coli Cultures and Purification of RecombinantAntibodies and Antigens

A shake flask inoculum is generated overnight from a −70° C. cell bankin an Innova 4330 incubator shaker (New Brunswick Scientific, Edison,N.J.) set at 37° C., 300 rpm. The inoculum is used to seed a 20 Lfermenter (Applikon, Foster City, Calif.) containing defined culturemedium (Pack et al., Bio/Technology 11, 1271-1277 (1993)) supplementedwith 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,Detroit, Mich.), 12.5 g/L glycerol and 10 mg/ml tetracycline. Thetemperature, pH and dissolved oxygen in the fermenter are controlled at26° C., 6.0-6.8 and 25% saturation, respectively. Foam is controlled byaddition of polypropylene glycol (Dow, Midland, Mich.). Glycerol isadded to the fermenter in a fed-batch mode. Fab expression is induced byaddition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L during thelate logarithmic growth phase. Cell density is measured by opticaldensity at 600 nm in an UV-1201 spectrophotometer (Shimadzu, Columbia,Md.). Final Fab concentrations are typically 100-500 mg/L. Following runtermination and adjustment of pH to 6.0, the culture is passed twicethrough an M-210B-EH Microfluidizer (Microfluidics, Newton, Mass.) at17000 psi. The high pressure homogenization of the cells releases theFab into the culture supernatant.

The first step in purification is expanded bed immobilized metalaffinity chromatography (EB-IMAC). Streamline Chelating resin(Pharmacia, Piscataway, N.J.) is charged with 0.1 M NiCl₂. It is thenexpanded and equilibrated in 50 mM acetate, 200 mM NaCl, 10 mMimidazole, 0.01% NaN₃, pH 6.0 buffer flowing in the upward direction. Astock solution is used to bring the culture homogenate to 10 mMimidazole, following which, it is diluted two-fold or higher inequilibration buffer to reduce the wet solids content to less than 5% byweight. It is then loaded onto the Streamline column flowing in theupward direction at a superficial velocity of 300 cm/hr. The cell debrispasses through unhindered, but the Fab is captured by means of the highaffinity interaction between nickel and the hexahistidine tag on the Fabheavy chain. After washing, the expanded bed is converted to a packedbed and the Fab is eluted with 20 mM borate, 150 mM NaCl, 200 mMimidazole, 0.01% NaN₃, pH 8.0 buffer flowing in the downward direction.The second step in purification uses ion-exchange chromatography (IEC).Q Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) is equilibratedin 20 mM borate, 37.5 mM NaCl, 0.01% NaN₃, pH 8.0. The Fab elution poolfrom the EB-IMAC step is diluted four-fold in 20 mM borate, 0.01% NaN₃,pH 8.0 and loaded onto the IEC column. After washing, the Fab is elutedwith a 37.5-200 mM NaCl salt gradient. The elution fractions areevaluated for purity using an Xcell II SDS-PAGE system (Novex, SanDiego, Calif.) prior to pooling. Finally, the Fab pool is concentratedand diafiltered into 20 mM borate, 150 mM NaCl, 0.01% NaN₃, pH 8.0buffer for storage. This is achieved in a Sartocon Slice system fittedwith a 10,000 MWCO cassette (Sartorius, Bohemia, N.Y.). The finalpurification yields are typically 50%. The concentration of the purifiedFab is measured by UV absorbance at 280 nm, assuming an absorbance of1.6 for a 1 mg/mL solution.

Expression of Antigen or Antibodies in Shake Flasks and Purification

A shake flask inoculum is generated overnight from a −70° C. cell bankin an incubator shaker set at 37° C., 300 rpm. The cells are cultured ina defined medium described above. The inoculum is used to seed a 2 LTunair shake flask (Shelton Scientific, Shelton, Conn.) which is grownat 37° C., 300 rpm. Expression is induced by addition of L(+)-arabinoseto 2 g/L during the logarithmic growth phase, following which, the flaskis maintained at 23° C., 300 rpm. Following batch termination, theculture is passed through an M-110Y Microfluidizer (Microfluidics,Newton, Mass.) at 17000 psi. The homogenate is clarified in a J2-21centrifuge (Beckman, Fullerton, Calif.).

Purification employs immobilized metal affinity chromatography.Chelating Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) ischarged with 0.1 M NiCl₂ and equilibrated in 20 mM borate, 150 mM NaCl,10 mM imidazole, 0.01% NaN₃, pH 8.0 buffer. A stock solution is used tobring the culture supernatant to 10 mM imidazole. The culturesupernatant is then mixed with the resin and incubated in the incubatorshaker set at room temperature, 150-200 rpm. The antigen is captured bymeans of the high affinity interaction between nickel and thehexahistidine tag on the antigen. The culture supernatant and resinmixture is poured into a chromatography column. After washing, theantigen is eluted with 20 mM borate, 150 mM NaCl, 200 mM imidazole,0.01% NaN₃, pH 8.0 buffer. The antigen pool is concentrated in a stirredcell fitted with a 10,000 MWCO membrane (Amicon, Beverly, Mass.). It isthen dialysed overnight into 20 mM borate, 150 mM NaCl, 0.01% NaN₃, pH8.0 for storage, using 12-14,000 MWCO dialysis tubing. The purifiedantigen is evaluated for purity by SDS-PAGE analysis. A yield of 150 mgof purified antigen per liter of shake flask culture is expected. Theconcentration of the C. difficile glutamate dehydrogenase antigen isbased on UV absorbance at 280 nm, assuming an absorbance of 1.48 for a 1mg/mL solution. Antibody shake flask expression and purification is doneas described for antigen.

Example 21 Preparation of 7F11 Monoclonal Antibody Synthesis ofAcetylthiopropionic Acid

To a stirred solution of 3-mercaptopropionic acid (7 ml, 0.08 moles) andimidazole (5.4 g, 0.08 moles) in tetrahydrofuran (THF, 700 ml) was addeddropwise over 15 min, under argon, a solution of 1-acetylimidazole (9.6g, 0.087 moles) in THF (100 ml). The solution was allowed to stir afurther 3 hr at room temperature after which time the THF was removed invacuo. The residue was treated with ice-cold water (18 ml) and theresulting solution acidified with ice-cold concentrated HCl (14.5 ml) topH 1.5-2. The mixture was extracted with water (2×50 ml), dried overmagnesium sulfate and evaporated. The residual crude yellow oily solidproduct (10.5 g) was recrystallized from chloroform-hexane to afford 4.8g (41% yield) acetylthiopropionic acid as a white solid with a meltingpoint of 44-45° C.

Decapeptide Derivatives

The decapeptide (SEQ ID NO:1), YPYDVPDYAS, (Chiron Mimotopes PeptideSystems, San Diego, Calif.) was dissolved (0.3 g) in dry DMF (5.4 mL) ina round bottom flask under argon with moderate stirring. Imidazole (0.02g) was added to the stirring solution. Separately, acetylthiopropionicacid (0.041 g) was dissolved in 0.55 mL of dry DMF in a round bottomflask with stirring and 0.056 g of 1,1′-carbonyldiimidazole (AldrichChemical Co., Milwaukee, Wis.) was added to the stirring solution. Theflask was sealed under argon and stirred for at least 30 min at roomtemperature. This solution was added to the decapeptide solution and thereaction mixture was stirred for at least six hr at room temperaturebefore the solvent was removed in vacuo. The residue in the flask wastriturated twice using 10 mL of diethyl ether each time and the etherwas decanted. Methylene chloride (20 mL) was added to the residue in theflask and the solid was scraped from the flask and filtered using a finefritted Buchner funnel. The solid was washed with an additional 20 mL ofmethylene chloride and the Buchner funnel was dried under vacuum. Inorder to hydrolyze the derivative to generate a free thiol, it wasdissolved in 70% DMF and 1 N potassium hydroxide was added to a finalconcentration of 0.2 N while mixing vigorously. The derivative solutionwas allowed to stand for 5 min at room temperature prior toneutralization of the solution by the addition of a solution containing0.5 M potassium phosphate, 0.1 M borate, pH 7.0, to which concentratedhydrochloric acid has been added to a final concentration of 1 M. Thethiol concentration of the hydrolyzed decapeptide derivative wasdetermined by diluting 10 μL of the solution into 990 μL of a solutioncontaining 0.25 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, AldrichChemical Co., Milwaukee Wis.) and 0.2 M potassium borate, pH 8.0. Thethiol concentration in mM units was equal to the A₄₁₂(100/13.76).

Preparation of Conjugates of Decapeptide Derivative with Keyhole LimpetHemocyanin and Bovine Serum Albumin

Keyhole limpet hemocyanin (KLH, 6 ml of 14 mg/ml, Calbiochem, San Diego,Calif.) was reacted with sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SULFO-SMCC) by adding 15mg of SULFO-SMCC and maintaining the pH between 7 and 7.5 with 1Npotassium hydroxide over a period of one hr at room temperature whilestirring. The protein was separated from the unreacted SULFO-SMCC by gelfiltration chromatography in 0.1 M potassium phosphate, 0.02 M potassiumborate, and 0.15 M sodium chloride, pH 7.0, and 24 ml of KLH-maleimidewas collected at a concentration of 3.1 mg/ml. The hydrolyzeddecapeptide derivative was separately added to portions of theKLH-maleimide in substantial molar excess over the estimated maleimideamounts present and the solution was stirred for 4 hr at 4° C. and theneach was dialyzed against 3 volumes of one liter of pyrogen-freephosphate-buffered saline, pH7.4, prior to immunization.

Bovine serum albumin (BSA, 3.5 ml of 20 mg/ml) was reacted with SMCC byadding a solution of 6.7 mg of SMCC in 0.3 ml acetonitrile and stirringthe solution for one hr at room temperature while maintaining the pHbetween 7 and 7.5 with 1N potassium hydroxide. The protein was separatedfrom unreacted materials by gel filtration chromatography in 0.1 Mpotassium phosphate, 0.02 M potassium borate, 0.15 M sodium chloride, pH7.0. The hydrolyzed decapeptide derivative was separately added toportions of the BSA-maleimide in substantial molar excess over theestimated maleimide amounts present and the solution was stirred for 4hr at 4° C. The solutions were used to coat microtiter plates for thedetection of antibodies that bound to the decapeptide derivative bystandard techniques.

Production and Primary Selection of Monoclonal Antibodies

Immunization of Balb/c mice was performed according to the method of Liuet al. Clin Chem 25, 527-538 (1987). Fusions of spleen cells withSP2/0-Ag 14 myeloma cells, propagation of hybridomas, and cloning wereperformed by standard techniques. Selection of hybridomas for furthercloning began with culture supernatant at the 96-well stage. A standardELISA procedure was performed with a BSA conjugate of decapeptidederivative adsorbed to the ELISA plate. Typically, a single fusion wasplated out in twenty plates and approximately 10-20 wells per plate werepositive by the ELISA assay. At this stage, a secondary selection couldbe performed if antibodies to the SMCC part of the linking arm were tobe eliminated from further consideration. An ELISA assay using BSAderivatized with SMCC but not linked to the decapeptide derivativeidentified which of the positive clones that bound the BSA conjugateswere actually binding the SMCC-BSA. The antibodies specific for SMCC-BSAmay be eliminated at this step. Monoclonal antibody 7F11, specific forthe decapeptide derivative, was produced and selected by this process.

Example 22 Preparation of 7F11 Magnetic Latex

MAG/CM-BSA

To 6 mL of 5% magnetic latex (MAG/CM, 740 μm 5.0%, Seradyn,Indianapolis, Ind.) was added 21 mL of water followed by 3 mL of 600 mM2-(4-morpholino)-ethane sulfonic acid, pH 5.9 (MES, Fisher Scientific,Pittsburgh, Pa.). Homocysteine thiolactone hydrochloride (HCTL, 480 mg,Aldrich Chemical Co., Milwaukee, Wis.) and1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDAC, 660 mg, AldrichChemical Co., Milwaukee, Wis.) were added in succession, and thereaction mixture was rocked at room temperature for 2 h. The derivatizedmagnetic latex was washed 3 times with 30 mL of water (with magnet as inExample 14) using probe sonication to resuspend the particles. Thewashed particles were resuspended in 30 mL of water. Three mL of asolution containing sodium hydroxide (2M) and EDTA (1 mM) was added tothe magnetic latex-HCTL suspension, and the reaction proceeded at roomtemperature for 5 min. The pH was adjusted to 6.9 with 6.45 mL of 1 Mhydrochloric acid in 500 mM sodium phosphate, 100 mM sodium borate. Thehydrolyzed magnetic latex-HCTL was separated from the supernate with theaid of a magnet, and then resuspended in 33 mL of 50 mM sodiumphosphate, 10 mM sodium borate, 0.1 mM EDTA, pH 7.0. The magnetic latexsuspension was then added to 2 mL of 36 mg mL⁻¹ BSA-SMCC (made asdescribed in Example 21 with a 5-fold molar excess of SMCC over BSA),and the reaction mixture was rocked overnight at room temperature.N-Hydroxyethylmaleimide (NHEM, 0.42 mL of 500 mM, Organix Inc., Woburn,Mass.) was added to cap any remaining thiols for 30 min. After 30 min,the magnetic latex-BSA was washed twice with 30 mL of 50 mM potassiumphosphate, 10 mM potassium borate, 150 mM sodium chloride, pH 7.0(50/10/150) and twice with 30 mL of 10 mM potassium phosphate, 2 mMpotassium borate, 200 mM sodium thiocyanate, pH 7.0 (10/2/200). Themagnetic latex-BSA was resuspended in 30 mL of 10/2/200.

7F11-SH (1:5)

To a solution of 7F11 (3.8 mL of 5.85 mg mL-1) was added 18 μL of SPDP(40 mM in acetonitrile). The reaction proceeded at room temperature for90 min after which taurine (Aldrich Chemical Co., Milwaukee, Wis.) wasadded to a final concentration of 20 mM. Fifteen min later DTT was addedto a final concentration of 2 mM, and the reduction reaction proceededat room temperature for 30 min. The 7F11-SH was purified on G-50 (40 mL)that was eluted with 50/10/150 plus 0.1 mM EDTA. The pool of purified7F11-SH was reserved for coupling to the MAG/CM-BSA-SMCC.

MAG/CM-BSA-7F11

SMCC (10 mg) was dissolved in 0.5 mL of dry dimethylformamide (AldrichChemical Co., Milwaukee, Wis.), and this solution was added to themagnetic latex-BSA suspension. The reaction proceeded at roomtemperature with gentle rocking for 2 h. Taurine was added to a finalconcentration of 20 mM. After 20 min the magnetic latex-BSA-SMCC wasseparated from the supernate with the aid of a magnet and thenresuspended in 10/2/200 (20 mL) with probe sonication. The magneticlatex was purified on a column of Superflow-6 (240 mL, SterogeneBioseparations Inc., Carlsbad, Calif.) that was eluted with 10/2/200.The buffer was removed, and to the magnetic latex cake was added 30 mLof 0.7 mg mL⁻¹ 7F11-SH. The reaction mixture was rocked overnight atroom temperature. After 20 hr the reaction was quenched withmercaptoethanol (2 mM, Aldrich Chemical Co., Milwaukee, Wis.) followedby NHEM (6 mM). The MAG/CM-7F11 was washed with 10/2/200 followed by50/10/150. The magnetic latex was then resuspended in 30 mL of50/10/150.

TABLE 3 PCR and sequencing Primer Sequence (SEQ ID NOS:2-19) A 5′(CACTCAACCCTATCTATTAATGTGGAATTCAAATGGACGAAGCAGGGATTC) B- 5′(GTAGGCAATAGGTATTTCATCGTTTCACTCCATCCAAA) C- 5′ (TCCGTGCCGGTTGTGAAG) D-5′ (TACGCGAGGCTTGTCAGT) E- 5′ (TTCATCACTACGGTCGTC) F- 5′(GACGGCAATGTCTGATGC) G- 5′ (GATATCAACGTTTATCTAATCAGGCCATGGCTGGTTGGGCAG)H- 5′ (GGCATCCCAGGGTCACCATG) I- 5′ (TCGCTGCCCAACCAGCCATG) J- 5′(GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAATTAAGAAT       CCCTGGGCACAATTTTC) K- 5′(AGAGCTGCAGAATTCAGCTGATCATCTCACCAATAAAAAACGCCCGGCGGCA       ACCGAGCGTTCTGAACAAATGGACGAAGCAGGGATTC) L- 5′(TCTCTCCAAGGAAGCTTAAAAAAAAGCCCGCTCATTAGGCGGGCTAGCTTAATCAATCATGCATCGTTTCACTCCATCCAAAAAAAC) M- 5′(ACAGGTACGAAGCTTATCGATGATAAGCTGTCAAACCAAGGAGCTTAAAAAA AAGCCCGCTCATTAGGC)N- 5′ (ACCCGTTTTTTTGGATGGAGTGAAACGATGCATTACCTATTGCCTACGGCA) O- 5′(GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAATTAAGAAGC       GTAGTAGTCCGGAACGTC) P- 5′(GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAATTAGTGAT       GGTGATGGTGATGACAATCCCTG) Q 5′(ACCCGTTTTTTTGGATGGAGTGAAACGATGTCAGGAAAAGATG TAAATGTC) R- 5′(GTGATAAACTACCGCATTAAAGCTTATCGA TGATAAGCTGTCAATTAGTGATGGTGATGGTGATGGTACCATCCTCTTAATTTCATAGC) Note: restriction enzyme sites arein bold type. GAATTC = EcoRI CCATGG = NcoI AAGCTT = HindIII

85 10 amino acids amino acid single linear 1 Tyr Pro Tyr Asp Val Pro AspTyr Ala Ser 1 5 10 51 base pairs nucleic acid single linear cDNA 2CACTCAACCC TATCTATTAA TGTGGAATTC AAATGGACGA AGCAGGGATT C 51 38 basepairs nucleic acid single linear cDNA 3 GTAGGCAATA GGTATTTCAT CGTTTCACTCCATCCAAA 38 18 base pairs nucleic acid single linear cDNA 4 TCCGTGCCGGTTGTGAAG 18 18 base pairs nucleic acid single linear cDNA 5 TACGCGAGGCTTGTCAGT 18 18 base pairs nucleic acid single linear cDNA 6 TTCATCACTACGGTCGTC 18 18 base pairs nucleic acid single linear cDNA 7 GACGGCAATGTCTGATGC 18 42 base pairs nucleic acid single linear cDNA 8 GATATCAACGTTTATCTAAT CAGGCCATGG CTGGTTGGGC AG 42 20 base pairs nucleic acid singlelinear cDNA 9 GGCATCCCAG GGTCACCATG 20 20 base pairs nucleic acid singlelinear cDNA 10 TCGCTGCCCA ACCAGCCATG 20 69 base pairs nucleic acidsingle linear cDNA 11 GTGATAAACT ACCGCATTAA AGCTTATCGA TGATAAGCTGTCAATTAAGA ATCCCTGGGC 60 ACAATTTTC 69 89 base pairs nucleic acid singlelinear cDNA 12 AGAGCTGCAG AATTCAGCTG ATCATCTCAC CAATAAAAAA CGCCCGGCGGCAACCGAGCG 60 TTCTGAACAA ATGGACGAAG CAGGGATTC 89 87 base pairs nucleicacid single linear cDNA 13 TCTCTCCAAG GAAGCTTAAA AAAAAGCCCG CTCATTAGGCGGGCTAGCTT AATCAATCAT 60 GCATCGTTTC ACTCCATCCA AAAAAAC 87 69 base pairsnucleic acid single linear cDNA 14 ACAGGTACGA AGCTTATCGA TGATAAGCTGTCAAACCAAG GAGCTTAAAA AAAAGCCCGC 60 TCATTAGGC 69 51 base pairs nucleicacid single linear cDNA 15 ACCCGTTTTT TTGGATGGAG TGAAACGATG CATTACCTATTGCCTACGGC A 51 71 base pairs nucleic acid single linear cDNA 16GTGATAAACT ACCGCATTAA AGCTTATCGA TGATAAGCTG TCAATTAAGA AGCGTAGTAG 60TCCGGAACGT C 71 75 base pairs nucleic acid single linear cDNA 17GTGATAAACT ACCGCATTAA AGCTTATCGA TGATAAGCTG TCAATTAGTG ATGGTGATGG 60TGATGACAAT CCCTG 75 51 base pairs nucleic acid single linear cDNA 18ACCCGTTTTT TTGGATGGAG TGAAACGATG TCAGGAAAAG ATGTAAATGT C 51 89 basepairs nucleic acid single linear cDNA 19 GTGATAAACT ACCGCATTAAAGCTTATCGA TGATAAGCTG TCAATTAGTG ATGGTGATGG 60 TGATGGTACC ATCCTCTTAATTTCATAGC 89 43 base pairs nucleic acid single linear cDNA 20 TTACCCCTGTGGCAAAAGCC GAGGTGCAGC TTCAGGAGTC AGG 43 43 base pairs nucleic acidsingle linear cDNA 21 TTACCCCTGT GGCAAAAGCC CAGGTCCAGC TGCAGCAGTC TGG 4343 base pairs nucleic acid single linear cDNA 22 TTACCCCTGT GGCAAAAGCCGAAGTGCAGC TGGTGGAGTC TGG 43 43 base pairs nucleic acid single linearcDNA 23 TTACCCCTGT GGCAAAAGCC GAGGTGAAGC TGGTGGAATC TGG 43 43 base pairsnucleic acid single linear cDNA 24 TTACCCCTGT GGCAAAAGCC CAGGTGCAGCTGAAGGAGTC AGG 43 43 base pairs nucleic acid single linear cDNA 25TTACCCCTGT GGCAAAAGCC CAGGTTACGC TGAAAGAGTC TGG 43 43 base pairs nucleicacid single linear cDNA 26 TTACCCCTGT GGCAAAAGCC GAGGTGAAGC TGGATGAGACTGG 43 43 base pairs nucleic acid single linear cDNA 27 TTACCCCTGTGGCAAAAGCC GAGGTAAAGC TTCTCGAGTC TGG 43 43 base pairs nucleic acidsingle linear cDNA 28 TTACCCCTGT GGCAAAAGCC GAAATGAGAC TGGTGGAATC TGG 4343 base pairs nucleic acid single linear cDNA 29 TTACCCCTGT GGCAAAAGCCGAAGTGAAGC TGGTGGAGTC TGA 43 43 base pairs nucleic acid single linearcDNA 30 TTACCCCTGT GGCAAAAGCC CAGGTTCAGC TGCAACAGTC TGA 43 43 base pairsnucleic acid single linear cDNA 31 TTACCCCTGT GGCAAAAGCC GAGATCCAGCTGCAGCAGTC TGG 43 43 base pairs nucleic acid single linear cDNA 32TTACCCCTGT GGCAAAAGCC GAAGTGATGC TGGTGGAGTC TGG 43 43 base pairs nucleicacid single linear cDNA 33 TTACCCCTGT GGCAAAAGCC GAGGTGCAGC CTGTTGAGTCTGG 43 43 base pairs nucleic acid single linear cDNA 34 TTACCCCTGTGGCAAAAGCC GACGTGAAGC ATATGGAGTC TGG 43 43 base pairs nucleic acidsingle linear cDNA 35 TTACCCCTGT GGCAAAAGCC GAAGTGAAGC TTGAGGAGTC TGG 4343 base pairs nucleic acid single linear cDNA 36 TTACCCCTGT GGCAAAAGCCGAGGTCCAGC TTCAGCAGTC AGG 43 43 base pairs nucleic acid single linearcDNA 37 TTACCCCTGT GGCAAAAGCC CAGGTCCAGC TGCAGCAGTC TAG 43 43 base pairsnucleic acid single linear cDNA 38 TTACCCCTGT GGCAAAAGCC CAGGTCCAGCTGCAGCAGTC TCG 43 43 base pairs nucleic acid single linear cDNA 39TTACCCCTGT GGCAAAAGCC GAGGTTCAGC TGCAGCAGTC TGT 43 43 base pairs nucleicacid single linear cDNA 40 TTACCCCTGT GGCAAAAGCC CAGGTCCAAC TGCAGCAGCCTGG 43 43 base pairs nucleic acid single linear cDNA 41 TTACCCCTGTGGCAAAAGCC GAGGTTCAGC TGCAGCAGTC TGG 43 43 base pairs nucleic acidsingle linear cDNA 42 TTACCCCTGT GGCAAAAGCC GAGGTCCAGC TGCAACAATC TGG 4343 base pairs nucleic acid single linear cDNA 43 TTACCCCTGT GGCAAAAGCCCAGGTCCACG TGAAGCAGTC TGG 43 43 base pairs nucleic acid single linearcDNA 44 TTACCCCTGT GGCAAAAGCC GATGTGCAGC TTCAGGAGTC GGG 43 43 base pairsnucleic acid single linear cDNA 45 TTACCCCTGT GGCAAAAGCC CAAGTTACTCTAAAAGAGTC TGG 43 43 base pairs nucleic acid single linear cDNA 46TTACCCCTGT GGCAAAAGCC GAAGTGCAGC TGTTGGAGAC TGG 43 43 base pairs nucleicacid single linear cDNA 47 TTACCCCTGT GGCAAAAGCC CAGATCCAGT TGGTGCAATCTGG 43 43 base pairs nucleic acid single linear cDNA 48 TTACCCCTGTGGCAAAAGCC GATGTGAACT TGGAAGTGTC TGG 43 43 base pairs nucleic acidsingle linear cDNA 49 TTACCCCTGT GGCAAAAGCC CAGGCTTATC TACAGCAGTC TGG 4343 base pairs nucleic acid single linear cDNA 50 TTACCCCTGT GGCAAAAGCCCAGGTCCAAG TGCAGCAGCC TGG 43 43 base pairs nucleic acid single linearcDNA 51 TTACCCCTGT GGCAAAAGCC GAAGTGCAGC TGGTGGAGAC TGC 43 43 base pairsnucleic acid single linear cDNA 52 TTACCCCTGT GGCAAAAGCC GACGTGCAGGTGGTGGAGTC TGG 43 43 base pairs nucleic acid single linear cDNA 53CTGCCCAACC AGCCATGGCC GATGTTTTGA TGACCCAAAC TCC 43 43 base pairs nucleicacid single linear cDNA 54 CTGCCCAACC AGCCATGGCC GACATCCAGA TGACCCAGTCTCC 43 43 base pairs nucleic acid single linear cDNA 55 CTGCCCAACCAGCCATGGCC GATATCCAGA TGACACAGAC TAC 43 43 base pairs nucleic acidsingle linear cDNA 56 CTGCCCAACC AGCCATGGCC GACATTGTGA TGACCCAGTC TCC 4343 base pairs nucleic acid single linear cDNA 57 CTGCCCAACC AGCCATGGCCAACATTGTGC TGACCCAATC TCC 43 43 base pairs nucleic acid single linearcDNA 58 CTGCCCAACC AGCCATGGCC GATGTTGTGA TGACCCAAAC TCC 43 43 base pairsnucleic acid single linear cDNA 59 CTGCCCAACC AGCCATGGCC GAAATTGTGCTCACCCAGTC TCC 43 43 base pairs nucleic acid single linear cDNA 60CTGCCCAACC AGCCATGGCC AGTATTGTGA TGACCCAGAC TCC 43 43 base pairs nucleicacid single linear cDNA 61 CTGCCCAACC AGCCATGGCC GATATTGTGC TAACTCAGTCTCC 43 43 base pairs nucleic acid single linear cDNA 62 CTGCCCAACCAGCCATGGCC CAAATTGTTC TCACCCAGTC TCC 43 43 base pairs nucleic acidsingle linear cDNA 63 CTGCCCAACC AGCCATGGCC GACATTCAGC TGACCCAGTC TCC 4343 base pairs nucleic acid single linear cDNA 64 CTGCCCAACC AGCCATGGCCGATATTGTGA TGACCCAGGC TGC 43 43 base pairs nucleic acid single linearcDNA 65 CTGCCCAACC AGCCATGGCC GACCTTGTGC TGACACAGTC TCC 43 43 base pairsnucleic acid single linear cDNA 66 CTGCCCAACC AGCCATGGCC GAAAATGTGCTCACCCAGTC TCC 43 43 base pairs nucleic acid single linear cDNA 67CTGCCCAACC AGCCATGGCC GAAACAACTG TGACCCAGTC TCC 43 43 base pairs nucleicacid single linear cDNA 68 CTGCCCAACC AGCCATGGCC GATGCTGTGA TGACCCAGATTCC 43 43 base pairs nucleic acid single linear cDNA 69 CTGCCCAACCAGCCATGGCC GACATCTTGC TGACTCAGTC TCC 43 43 base pairs nucleic acidsingle linear cDNA 70 CTGCCCAACC AGCCATGGCC GATGTTGTGA TAACTCAGGA TGA 4343 base pairs nucleic acid single linear cDNA 71 CTGCCCAACC AGCCATGGCCGATGTTGTGG TGACTCAAAC TCC 43 43 base pairs nucleic acid single linearcDNA 72 CTGCCCAACC AGCCATGGCC AACATTGTGA TGGCCTGGTC TCC 43 43 base pairsnucleic acid single linear cDNA 73 CTGCCCAACC AGCCATGGCC TCATTATTGCAGGTGCTTGT GGG 43 43 base pairs nucleic acid single linear cDNA 74CTGCCCAACC AGCCATGGCC GATATTGTGA TAACCCAGGA TGA 43 43 base pairs nucleicacid single linear cDNA 75 CTGCCCAACC AGCCATGGCC GACATTGTGA TGACCCAGTCTCA 43 43 base pairs nucleic acid single linear cDNA 76 CTGCCCAACCAGCCATGGCC GAAATGGTTC TCACCCAGTC TCC 43 43 base pairs nucleic acidsingle linear cDNA 77 CTGCCCAACC AGCCATGGCC GATGTTGTGC TGACCCAAAC TCC 4343 base pairs nucleic acid single linear cDNA 78 CTGCCCAACC AGCCATGGCCGACGTTGTGA TGTCACAGTC TCC 43 43 base pairs nucleic acid single linearcDNA 79 CTGCCCAACC AGCCATGGCC GACATTGTGA CGTCACAGTC TCC 43 43 base pairsnucleic acid single linear cDNA 80 CTGCCCAACC AGCCATGGCC CAAGTTGTTCTCACCCAGTC TCC 43 43 base pairs nucleic acid single linear cDNA 81CTGCCCAACC AGCCATGGCC GACGTCCAGA TAACCCAGTC TCC 43 20 base pairs nucleicacid single linear cDNA 82 GATGGGGGTG TCGTTTTGGC 20 20 base pairsnucleic acid single linear cDNA 83 ACAGTTGGTG CAGCATCAGC 20 70 basepairs nucleic acid single linear cDNA 84 TATTTCCAGC TTGGTCCCTCTAGAGTTAAC GATATCAACG TTTATCTAAT CAGCAAGAGA 60 TGGAGGCTTG 70 70 basepairs nucleic acid single linear cDNA 85 TGAGGTTCCT TGACCCCACTGCAGAGTACT AGGCCTCTGA GCTACTCAGT TAGGTGATTG 60 AGTAGCCAGT 70

What is claimed is:
 1. A method of producing a polypeptide library having affinity for a target, comprising: providing a library of viral particles, wherein a member comprises a viral particle capable of displaying from its outersurface a fusion protein comprising a viral coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members, and wherein the library of viral particles has been enriched by affinity selection; subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules; and introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms.
 2. The method of claim 1, further comprising releasing the polypeptides from the host.
 3. The method of claim 1, wherein the polypeptide to be screened comprises an antibody heavy or light chain variable domain, and in at least some of the members of the library of viral particles, the heavy or light chain variable domain is respectively associated with a binding partner comprising a partner light or heavy chain antibody variable domain to form a Fab fragment.
 4. The method of claim 3, wherein both the antibody heavy or light chain variable domain and the partner light or heavy chain variable domain are encoded by the genome of the viral particle, and the subcloning step comprises subcloning DNA encoding both the heavy or light chain variable domain and the partner light or heavy chain variable domain from members of the viral particle library to the expression vector, wherein antibodies are expressed in the host cells and released from the host cells to form an antibody library.
 5. The method of claim 4, wherein the host cells are procaryotic and the antibody heavy or light chain variable domain and the partner light or heavy chain variable domain are expressed from the same promoter in the expression vector as a polycistronic message.
 6. The method of claim 5 wherein the promoter is inducible.
 7. The method of claim 6, wherein the promoter is an arabinose promoter.
 8. The method of claim 4, wherein the subcloning comprises: amplifying in vitro a segment of DNA encoding the antibody heavy or light chain variable domain and the partner light or heavy chain variable domain; digesting the amplified DNA with T4 DNA polymerase to create single stranded termini of the amplified DNA; digesting linearized expression vector with T4 DNA polymerase to create single stranded termini of the expression vector; annealing the linearized expression vector with the amplified DNA.
 9. The method of claim 4, further comprising applying positive selection for expression vector having acquired an insert comprising DNA encoding the antibody heavy or light chain variable domain and the partner light or heavy chain variable domain.
 10. The method of claim 9, wherein the expression vector comprises a selection marker lacking a terminal segment which prevents expression of the selection marker and acquisition of the insert restores the terminal segment whereby the selection marker is expressed.
 11. The method of claim 1, further comprising contacting the library of at least ten different polypeptides with a tissue sample suspected of containing the target molecule and determining whether at least one of the polypeptides specifically binds to the target.
 12. The method of claim 1, further comprising providing a primary library of replicable genetic packages, wherein a member comprises a replicable genetic package capable of displaying from its outersurface a fusion protein comprising an outersurface protein of the package and a displayed polypeptide, the fusion protein being encoded by a segment of the genome of the package and the polypeptides varying between members; wherein the primary library encodes at least 100-fold more different polypeptides than the library of replicable genetic packages; contacting the primary library with the target molecule and separating replicable genetic packages bound to the target molecule from unbound replicable genetic packages; amplifying the bound replicable genetic packages to form a subprimary library; repeating the previous two steps until the subprimary library constitutes the library, wherein at least 95% of library members encode polypeptides have specific affinity for the same target molecule and no library member constitutes more than 50% of the total forms.
 13. The method of claim 1, wherein the target is a cellular receptor and the library of polypeptides block the cellular receptor.
 14. The method of claim 13, wherein the phage particle comprises a phagemid genome, which encodes the fusion protein.
 15. The method of claim 1, wherein the viral particle is a phage particle.
 16. The method of claim 15, wherein the phage coat protein is filamentous phage pIII or pVIII or a fragment of either of these sufficient to display the displayed polypeptide from the outersurface of the phase particle.
 17. A method of preparing a diagnostic kit containing a polypeptide library having affinity for a target, comprising: providing a library of viral particles, wherein a member comprises viral particle capable of displaying from its outersurface a fusion protein comprising a phage coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members, and wherein the library of viral particles has been enriched by affinity selection; subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules; introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms, and incorporating the library of polypeptides into a diagnostic kit.
 18. A method of preparing a diagnostic reagent comprising a polypeptide library having affinity for a target, comprising: providing a library of viral particles, wherein a member comprises viral particle capable of displaying from its outersurface a fusion protein comprising a phage coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members, and wherein the library of viral particles has been enriched by affinity selection; subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules; introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms, and formulating the library of polypeptides as a diagnostic reagent.
 19. A method of producing an antibody library having affinity for a target, comprising: providing a library of phage, wherein a member of the library comprises a phage capable of displaying from its outersurface an antibody comprising an antibody heavy chain variable domain complexed with an antibody light chain variable domain, wherein either the heavy or light chain variable domain is expressed as a fusion protein with a coat protein of the phage and either the heavy or light chain variable domain or both is/are encoded by the genome of the phage, and the heavy and/or light chain varies between members; subcloning a mixture of DNA molecules encoding the heavy and/or light chain variable domains from the phage library members into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules; introducing the modified forms of the expression vector into a host and expressing antibodies formed by the heavy and light chain variable domains of the phage library in the host, the antibodies being released from the host to form an antibody library of at least ten antibodies wherein at least 90% of modified forms of the expression vector encode antibodies with specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms.
 20. A method of preparing a therapeutic composition comprising a polypeptide library having affinity for a target, comprising: providing a library of viral particles, wherein a member comprises viral particle capable of displaying from its outersurface a fusion protein comprising a phage coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides vary between members, subcloning a mixture of DNA molecules encoding at least ten different polypeptides of the library of viral particles into multiple copies of an expression vector to produce modified forms of the expression vector; introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms, and formulating the library of polypeptides with a pharmaceutically acceptable carrier to form a therapeutic composition.
 21. A method of producing a polypeptide library having affinity for a target, comprising: providing a library of viral particles, wherein a member comprises a viral particle capable of displaying from its outersurface a fusion protein comprising a viral coat protein and a displayed polypeptide, the fusion protein encoded by a genome of the viral particle and the polypeptides varying between members, contacting the library members with a receptor that specific affinity for a peptide sequence of the fusion protein that is the same in each library member and isolating library members bound to the receptor in immobilized form; contacting the isolated library members with a target molecule, and isolating library members bound to the target molecule in immobilized form; subcloning a mixture of DNA molecules encoding at least ten different polypeptides from the isolated library members that bind to the same target molecule; into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules; and introducing the modified forms of the expression vector into a host and expressing the polypeptides in the host, wherein a library of at least ten different polypeptides are expressed, at least 90% of modified forms of the expression vector encode polypeptides that, optionally in association with a binding partner, have specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms.
 22. A method of producing an antibody library having affinity for a target, comprising: providing a library of phage, wherein a member of the library comprises a phage capable of displaying from its outersurface an antibody comprising an antibody heavy chain variable domain complexed with an antibody light chain variable domain, wherein either the heavy or light chain variable domain is expressed as a fusion protein with a coat protein of the phage and either the heavy or light chain variable domain or both is/are encoded by the genome of the phage, and the heavy and/or light chain varies between members; contacting the library members with a receptor that has specific affinity for a peptide sequence of the fusion protein that is the same in each library member and isolating library members that bind to the receptor in immobilized form; contacting the isolated library members with a target molecule, and isolating library members that bind to the target molecule in immobilized form; subcloning a mixture of DNA molecules encoding the heavy and/or light chain variable domains from the isolated phage library members that bind to the same target molecule into multiple copies of an expression vector to produce modified forms of the expression vector by insertion of one of the DNA molecules; introducing the modified forms of the expression vector into a host and expressing antibodies formed by the heavy and light chain variable domains of the phage library in the host, the antibodies being released from the host to form an antibody library of at least ten antibodies wherein at least 90% of modified forms of the expression vector encode antibodies with specific affinity for the same target molecule and no modified form of the expression vector constitutes more than 50% of the total modified forms. 